Method for orthopoxvirus production and purification

ABSTRACT

The present invention relates to a method for producing and purifying a wild type, an attenuated and/or a recombinant  Orthopoxvirus . The present invention relates to a purified wild type, attenuated and/or recombinant  Orthopoxvirus  obtained by the method of the invention and to a pharmaceutical composition, preferably a vaccine, comprising said purified  Orthopoxvirus  for the treatment and/or the prevention a cancer, an infectious disease and/or an autoimmune disorder, and uses thereof The present invention also relates to the use of an immortalized avian cell line obtained from an avian cell belonging to the Anatidae family, in particular  Cairina moschata  immortalized avian cell lines comprising a nucleic acid sequence coding a telomerase reverse transcriptase (TERT) and optionally an E1A nucleic acid sequence, for the production of a wild type, attenuated and/or recombinant  Orthopoxvirus  according to the process of the invention

TECHNICAL FIELD

The present invention pertains to the field of virus production andpurification. In particular, the invention relates to a method forproducing and purifying a wild type, an attenuated and/or a recombinantOrthopoxvirus.

BACKGROUND OF THE INVENTION

Orthopoxviruses are complex enveloped viruses having a diametercomprised between 200 and 300 nm that distinguish them principally bytheir unusual morphology, their large DNA genome and their cytoplasmicsite of replication. The genome of several members of Orthopoxviruses,including the Copenhagen Vaccinia Virus (VV) strain (GOEBEL et al.,1990, Virol. 179, 247-266 and 517-563; JOHNSON et al., 1993, Virol. 196,381-401) and the modified Vaccinia Virus Ankara (MVA) strain (ANTOINE etal., 1998, Virol. 244, 365-396), have been mapped and sequenced. VV hasa double-stranded DNA genome of about 192 kb coding for about 200proteins of which approximately 100 are involved in virus assembly. MVAis a highly attenuated Vaccinia Virus strain generated by more than 500serial passages of the Ankara strain of Vaccinia Virus on chicken embryofibroblasts (MAYR et al., 1975, Infection 3, 6-16). The MVA virus wasdeposited before Collection Nationale de Cultures de Microorganismes(CNCM) under depositary N⁶⁰² I-721. Determination of the completesequence of the MVA genome and comparison with the Copenhagen VV genomeallows the precise identification of the alterations which occurred inthe viral genome and the definition of seven deletions (I to VII) andnumerous mutations leading to fragmented ORFs (Open Reading Frame)(ANTOINE et al., 1998, Virology 244, 365-396).

The use of Orthopoxviruses as vectors for the development of recombinantlive vaccines has been affected by safety concerns and regulations.Before using Orthopoxviruses for vaccination it is necessary to purifythe viruses. Available Orthopoxviruses production methods comprise thereplication of the virus in a cell line (e.g. HelaS3), in embryonatedeggs or in Chicken Embryo Fibroblasts (CEFs). After the replication ofthe virus, the culture media is discard, the cells are lysed and theOrthopoxviruses released from the cells are purified by sucrose cushioncentrifugation (KOTWAL and ABRAHAM; poxvirus growth, Purification andtittering in Vaccinia Virus and Poxvirology, 2004, 101-108, Humana PressInc., Totowa; N.J.; USA).

International patent application WO 07/147528 describes a method forproducing a wild type, an attenuated and/or a recombinant MVA with notargeted infection specificity, comprising preparing a culture ofpackaging cells (e.g. CEFs; cell lines), infecting said cell culture,culturing said infected cells, recovering the MVAs produced from theculture supernatant and/or the packaging cells, and further purifyingthe viruses by depth filtration, microfiltration and diafiltration. WO07/147528 specifies that when depth filtration, microfiltration anddiafiltration are performed, the use of nucleases and more particularly,the use of Benzonase® as nuclease is not necessary.

International patent application WO 08/138533 describes a method for thepurification of biologically active vaccinia virus, comprising loading asolid-phase matrix to which a ligand is attached with a vaccinia viruscontained in a liquid-phase culture, washing the matrix, and eluting thevirus.

Despite the description of several methods for Orthopoxvirus productionand purification, a need remains for alternative. The present inventionprovides such methods.

DISCLOSURE OF THE INVENTION

As used throughout in the entire application, an “Orthopoxvirus” refersto a variola virus; a Vaccinia Virus (VV) such as for instance theVaccinia virus strains: Elstree, Western Reserve, Wyeth, NYVAC, NYCBOH,Paris, Copenhagen; and their derivatives such as for instance a modifiedVaccinia Virus Ankara (MVA) in particular MVA 575 (ECACC V00120707) andMVA-BN (ECACC V00083008). According to the invention the Orthopoxviruscan be indifferently an immature virus (IV), an intracellular maturevirus (IMV), an intracellular enveloped virus (IEV), a cell-associatedenveloped virus (CEV) or an extracellular enveloped virus (EEV) (SMITHet al. (2002), J. Gen. Virol., 83, 2915-2931).

As used throughout the entire application, “a” and “an” are used in thesense that they mean “at least one”, “at least a first”, “one or more”or “a plurality” of the referenced components or steps, unless thecontext clearly dictates otherwise.

As used throughout the entire application, “and/or” wherever used hereinincludes the meaning of “and”, “or” and “all or any other combination ofthe elements connected by said term”.

As used throughout the entire application, “comprising” and “comprise”are intended to mean that the products, compositions and methods includethe referenced components or steps, but not excluding others.“Consisting essentially of” when used to define products, compositionsand methods, shall mean excluding other components or steps of anyessential significance. Thus, a composition consisting essentially ofthe recited components would not exclude trace contaminants andpharmaceutically acceptable carriers. “Consisting of” shall meanexcluding more than trace elements of other components or steps.

As used throughout the entire application, “about” or “approximately” asused herein means within 20%, preferably within 10%, and more preferablywithin 5% of a given value or range.

The present invention relates to a method (i.e. Method A) for producingand purifying a wild type, an attenuated and/or a recombinantOrthopoxvirus, comprising the following steps:

-   -   a) preparing a culture of packaging cells;    -   b) infecting the packaging cell culture with an Orthopoxvirus;    -   c) culturing the infected packaging cells until progeny        Orthopoxvirus is produced;    -   d) incubation in presence of one or more nucleases;    -   e) recovering the Orthopoxviruses from the culture supernatant        and/or the packaging cells;    -   f) adding monovalent salts to the Orthopoxviruses recovered in        step e) under suitable conditions to inhibit the nuclease(s)        activity and to avoid the adsorption of said Orthopoxviruses to        the anion exchange adsorbent in step g);    -   g) contacting the mixture obtained in step f) with an anion        exchange adsorbent under suitable conditions to allow the        capture of nucleic acids;    -   h) clarifying the mixture obtained in step g) under suitable        conditions to allow the withdrawal of the cellular debris;    -   i) washing of the anion exchange adsorbent with a solution        comprising monovalent salts under suitable conditions to recover        the remained Orthopoxviruses in the flow through;    -   j) concentrating the flow through obtained in step h) and the        flow through obtained in step i);    -   k) diafiltrating the fraction comprising the Orthopoxviruses        obtained in step j).

According to a preferred embodiment, the method according to theinvention is free from animal products (except the packaging cells). Asused throughout the entire application, <<animal products>> refer to anycompound or collection of compounds that was produced in or by an animalcell in a living organism.

According to a preferred embodiment, the method according to theinvention is suitable for an aseptic industrial-scale manufacturingprocess to ensure a full compliance with regulatory requirementsregarding sterility of vaccines.

As used throughout the entire application, “attenuated Orthopoxvirus”refers to any Orthopoxvirus that has been modified so that itspathogenicity in the intended subject is substantially reduced.Preferably, the Orthopoxvirus is attenuated to the point it isnonpathogenic from a clinical standpoint, i.e. that subjects exposed tothe Orthopoxvirus do not exhibit a statistically significant increasedlevel of pathology relative to control subjects. According to apreferred embodiment of the invention, the attenuated Orthopoxvirus isan attenuated VV or an attenuated MVA.

As used throughout the entire application, “recombinant Orthopoxvirus”refers to an Orthopoxvirus comprising an exogenous sequence inserted inits genome. As used herein, an exogenous sequence refers to a nucleicacid which is not naturally present in the parent virus.

As used throughout the entire application, “packaging cells” refers to acell which can be infected by the Orthopoxvirus to be produced. Thepackaging cell may be a primary cell, a recombinant cell and/or a cellline. For example, a recombinant cell which contains the elementsnecessary for the production of a recombinant virus which are lacking ina recombinant viral vector can be used.

According to a preferred embodiment of the invention, the packagingcells are immortalized cell lines. As used throughout the entireapplication, “immortalized cell lines” refer to cell lines thatproliferate in culture beyong the Hayflick limit.

According to the invention, the immortalized cell lines are preferablyobtained from an avian cell belonging to the Anatidae family or thePhasianidae family. Among Anatidae, cell belonging to the Cairina orAnas genus is particularly preferred. Even more preferably, theimmortalized avian cell lines belong to the Cairina moschata or to theAnas platyrhynchos species.

According to a more preferred embodiment of the invention, the packagingcells are immortalized avian cell lines obtained from an avian cellbelonging to the Anatidae family, and preferably from Cairina moschataspecie.

Preferred Cairina moschata immortalized avian cell lines according tothe invention, are Cairina moschata immortalized avian cell linescomprising a nucleic acid sequence coding a telomerase reversetranscriptase (TERT) covered by patent application WO 2007/077256. Areparticularly preferred, the following immortalized avian cell lines:

-   -   T3-17490 as deposited at the European Collection of Cell        Cultures (ECACC) under accession number 08060502 (see FIGS. 2, 3        and 4) or a derivative thereof;    -   T6-17490 as deposited at the European Collection of Cell        Cultures (ECACC) under accession number 08060501 (see FIGS. 5, 6        and 7) or a derivative thereof.

Other preferred Cairina moschata immortalized avian cell lines accordingto the invention, are Cairina moschata immortalized avian cell linescomprising an E1A nucleic acid sequence and a nucleic acid sequencecoding a telomerase reverse transcriptase (TERT) covered by patentapplication WO 2009/004016.

As used throughout the entire application, “derivative” of the depositedimmortalized avian cell lines refers to an immortalized avian cell linewhich comprises a nucleic acid sequence coding a “substance ofinterest”. As used herein, a “substance of interest” can include, but isnot limited to, a pharmaceutically active protein e.g. growth factors,growth regulators, antibodies, antigens, their derivatives useful forimmunization or vaccination and the like, interleukins, insulin,erythropoietin, G-CSF, GM-CSF, hPG-CSF, M-CSF, interferons(interferon-alpha, interferon-beta, interferon-gamma), blood clottingfactors (e.g. Factor VIII; Factor IX; tPA) or combinations thereof.

Other immortalized cell lines that may be used in the method of thepresent invention are:

-   -   DF1 cell line covered by patent U.S. Pat. No. 5,879,924, which        is a spontaneously immortalized chicken cell line derived from        10 day old East Lansing Line (ELL-0) eggs;    -   Ebx chicken cell line covered by patent application WO        2005/007840, which derives from embryonic stem cells by        progressive severance from growth factors and feeder layer;    -   DEC 99 cell line (Ivanov et al. Experimental Pathology and        Parasitology, 4/2000 Bulgarian Academy of Sciences), which is        duck embryo permanent cell line.

According to another preferred embodiment of the invention, thepackaging cells are primary cells. As used throughout the entireapplication, “primary cells” refer to cells that have been freshlyisolated from an animal or human tissue, organ or organism, wherein thecells are not able to continuously and indefinitely replicate anddivide. Usually, primary cells divide in cell culture less than 100times, often less than 50 times, often less than 25 times. Primary cellshave therefore not undergone an immortalized event. Primary cellsinclude but are not limited to animal fibroblasts or cord bloodlymphocytes.

According to a more preferred embodiment of the invention, the packagingcells are primary or secondary avian cells, and preferably chickenembryo fibroblasts (CEFs).

According to the present invention, the culture medium used for thepreparation of the culture of packaging cells (i.e. during step a)), theculture medium used for the infection of said cell culture with anOrthopoxvirus (i.e. during step b)) and the culture medium used for thecultivation of said infected packaging cells (i.e. during step c)) canbe the same or different.

According to a preferred embodiment, the cell culture media usedaccording to the invention are free from animal product. Many media freefrom animal product have been already described and some of them arecommercially available. For example 293 SFM II; 293-F Cells, SFMAdapted; 293-H Cells, SFM Adapted; 293fectin™ Transfection Reagent; CD293 AGT™; CD 293 Medium; FreeStyle™ 293 Expression System; FreeStyle™293 Medium; FreeStyle™ 293-F Cells, SFM Adapted; Adenovirus ExpressionMedium (AEM) Growth Medium for PER.C6® Cells; CD 293 AGT™; CD 293 Medium; COS-7L Cells, SFM Adapted; EPISERF® Medium; OptiPro™ SFM; VP-SFM;VP-SFM AGT™. (all available from Invitrogen) can be used as cell culturemedia in the process according to the invention. Cell culture media freefrom animal product used according to the invention can also behome-made media.

Step a) of preparation of a culture of packaging cells is well known tothe one skilled in the art.

When the packaging cells are immortalized cell lines, said immortalizedcell lines are cultured in the appropriate culture media. The methodsmay comprise growth adhering to surfaces, growth in suspension inpresence or not of (micro)carriers, or combinations thereof. Culturingcan be done for instance in dishes, roller bottles or in bioreactors,using batch, fed-batch, continuous systems, hollow fiber, and the like.In order to achieve large scale production of virus through cell cultureit is preferred in the art to have cells capable of growing insuspension in presence or not of (micro)carriers, and it is preferred tohave cells capable of being cultured in media free from animal product.Cell culture media used according to the invention are preferably freefrom animal product. Many media free from animal product has beenalready described and some of them are commercially available aspreviously described.

When the packaging cells are CEFs, said CEFs are preferably extractedfrom Specific Pathogen Free (SPF) eggs. SPF eggs are commerciallyavailable, for example from Charles River Laboratories (Wilmington,Mass., USA). Said eggs are preferably more than 9 days old, morepreferably between 10 and 14 days old and even more preferably are 12days old. Before the extraction of the embryo, the egg is preferablydisinfected. Many methods and products dedicated to the disinfection ofeggs are available in the prior art. Incubation in a formol solution(e.g. 2% formol, 1 min.) followed by a rinsing in 70% ethanol isparticularly preferred. The cells of the embryos are then dissociatedand purified. According to a preferred embodiment of the invention, thecells of the embryos are subjected to an enzymatic digestion step thatallows the destruction of the intercellular matrix. For this purpose,the use of enzymes able to digest the intercellular matrix isparticularly useful. Preferred enzyme according to the invention includebut are not limited to Trypsin, Collagenase, Pronase, Dispase,Hyaluronidase and Neuraminidase. The enzymes used for the preparation ofCEFs according to the invention are preferably of recombinant origin.The enzymes can be used alone or in combination. In a preferredembodiment of the invention dispase and tryspsin (e.g. TrypLE selectfrom Gibco™) are used in combination. The one skilled in the art is ableto determine the enzyme concentration, the temperature and the length ofincubation allowing an efficient separation of the cells. Thepreparation of the CEFs culture can further include a filtration stepand/or a centrifugation step in order to remove contaminants. Accordingto the invention, the primary CEFs obtained can also either be useddirectly or after one further cell passage as secondary CEFs. The CEFs(i.e. primary or secondary) are then cultivated in an appropriate cellculture medium. Cell culture media used according to the invention arepreferably free from animal product. Many media free from animal producthas been already described and some of them are commercially availableas previously described. According to the present invention, the CEFsare preferably cultivated in VP-SFM cell culture medium (Invitrogen).The CEFs are preferably cultivated for between 1 and 5 days, morepreferably between 1 and 2 days and even more preferably 2 days beforeinfection. The CEFs are preferably cultivated at a temperature comprisedbetween 30° C. and 36.5° C.

Step b) of infection of the packaging cell culture (prepared in step a))with an Orthopoxvirus is well known to the one skilled in the art. Asused throughout the entire application, “infection” refers to thetransfer of the viral nucleic acid to a cell, wherein the viral nucleicacid is replicated, viral proteins are synthesized, or new viralparticles assembled. The one skilled in the art is able to select themost appropriate packaging cell for the production of a specific virus.According to a preferred embodiment, the method according to theinvention comprises the use of CEFs or an immortalized avian cell linecovered by patent application W0 2007/077256 or W0 2009/004016 for theproduction of an Orthopoxvirus, and preferably a MVA or a VV. Step b) ofinfection of the packaging cell culture (prepared in step a)) with anOrthopoxvirus is performed in an appropriate cell culture medium whichcan be the same or different from the cell culture medium used for thepreparation of said packaging cell culture (i.e. during step a)). Cellculture media used according to the invention are preferably free fromanimal product. Many media free from animal product have been alreadydescribed and some of them are commercially available as previouslydescribed. When the packaging cells are CEFs, step b) of infection ofthe CEFs culture is performed in Basal Medium Eagle cell culture medium(Invitrogen). The cell culture medium is preferably seeded with betweenbetween 0.5 to 1.5 and more preferably between 1.1 and 1.3 and even morepreferably about 1.2 embryo/l of cell culture medium. In the specificembodiment where the Orthopoxvirus to produce is MVA, the MVA is seededin the cell culture vessel at a MOI which is preferably comprisedbetween 0.001 and 0.1, more preferably between 0.03 and 0.07 and evenmore preferably about 0.05. In another specific embodiment where theOrthopoxvirus to produce is VV, the VV is seeded in the cell culturevessel at a MOI which is preferably comprised between 0.0011 and 0.1,and more preferably about 0.0001.

Step c) of culture of the infected packaging cells (from step b)) untilprogeny Orthopoxvirus is produced, is well known to the one skilled inthe art.

When the packaging cells are CEFs, step c) of culture of the infectedCEFs is performed in an appropriate cell culture medium which can be thesame or different from the cell culture medium used for the preparationof said CEFs culture (i.e. during step a) and from the cell culturemedium used for the infection of said CEFs culture with an Orthopoxvirus(i.e. during step b)). Cell culture media used according to theinvention are preferably free from animal product. Many media free fromanimal product have been already described and some of them arecommercially available as previously described. According to the presentinvention, the culture of the infected CEFs is performed in Basal MediumEagle cell culture medium (Invitrogen). The infected CEFs are preferablycultivated for between 1 and 6 days, more preferably between 2 and 4days and even more preferably 3 days. The infected CEFs are preferablycultivated at a temperature which is lower than 37° C., preferablybetween 30° C. and 36.5° C.

Step d) of incubation in presence of one or more nucleases (i.e.endonuclease or exonucleases) is performed in order to degrade thenucleic acids (e.g. DNA; RNA) present in solution. Nucleases preferablyused according to the present invention are endonucleases. Endonucleasescan be classified based on their substrates as follows:deoxyribonucleases (DNases) which degrade DNA; ribonucleases (RNases)which degrade RNA; and endonucleases that degrade DNA and RNA.Endonucleases DNases include but are not limited to DNase I, DNase IIand endodeoxyribonuclease IV. Endonucleases RNases include but are notlimited to RNase I, RNase III, RNAse E, RNAse F and RNAse P.Endonucleases that degrade DNA and RNA include but are not limited toBenzonase®. In a preferred embodiment of the invention, step d) ofincubating the Orthopoxviruses produced in step c) is performed inpresence of Benzonase®. Benzonase® degrades nucleic acid (e.g. DNA; RNA)by hydrolyzing internal phosphodiester bonds between specificnucleotides. Upon complete digestion, all free nucleic acids (e.g. DNA;RNA) present in solution are reduced to 5′-monophosphate terminatedoligonucleotides which are 3 to 8 bases in length. Benzonaze® has noproteolytic activity. Benzonaze® used according to the present inventionis preferably pharmaceutically acceptable. Pharmaceutically acceptableBenzonaze® are commercially available (e.g. Eurogentec under thereference ME-0280-10; Merck under the reference e.g. 1.01653.0001).

Preferred conditions for the action of nuclease(s) according to theinvention are (as described in Example 1):

-   -   a pH comprised between 7.0 and 9.0, preferably between 7.5 and        8.5, and more preferably a pH of 8.0;    -   a concentration of cofactors selected from Mg²⁺ and Mn²⁺,        preferably Mg²⁺, in a range of 1 to 2 mM, and preferably 2 mM.

According to a preferred embodiment of the invention, the nuclease(s)is(are) incubated at a temperature comprised between 22° C. and 28° C.,preferably at a temperature comprised between 23° C. and 27° C., morepreferably at a temperature comprised between 24° C. and 26° C., andeven more preferably at a temperature of 25° C. (as described in Example1). In this present embodiment, the duration of step d) is preferablycomprised between 1 and 5 hours, and more preferably 2 hours (asdescribed in Example 1).

According to another preferred embodiment of the invention, thenuclease(s) is(are) incubated at a temperature comprised between 2° C.and 8° C., preferably at a temperature comprised between 3° C. and 7°C., more preferably at a temperature comprised between 4° C. and 6° C.,and even more preferably at a temperature of 5° C. In this otherembodiment, the duration of step d) is preferably comprised between 10and 20 hours, and more preferably 18 hours.

According to the invention, the concentration of nuclease(s) used instep d) is in a range of 5 to 100 U/ml, preferably in a range of 5 to 50U/ml, and more preferably 10 U/ml. As showed in FIG. 1, the use underthe same conditions of 10 U/ml Benzonase® leads surprisingly to anequivalent decrease of DNA concentration after 2 hours of treatment(temperature of 25° C.; 2 mM Mg²⁺; pH 8) compared with the use of 50U/ml Benzonase®.

According to a preferred embodiment of the invention, step d) furthercomprises the addition of one or more detergents. Detergents include butare not limited to Tween, Triton X-100 (nonaethylene glycol octyl phenolether), saponin, SDS, Brij 96, Polido-canol, N-octyl R-D-glucopyranosideand sodium carbonate. Preferred detergent used in step d) is Tween.Tween include but is not limited to Tween 20 (Polyoxyethylene SorbitanMonolaurate), Tween 80 (Polyoxyethylene Sorbitan Monooleate) and Tween85 (Polyoxyethylene Sorbitan Trioleate). Preferred Tween used in step d)is Tween 80. According to the invention, the concentration ofdetergent(s) used in step d) is in a range of 10 to 100 μg/L, preferablyin a range of 10 to 55 μg/L.

The Orthopoxviruses produced and previously treated by nuclease(s) arethen recovered from the culture supernatant and/or the packaging cells.

When the Orthopoxviruses are recovered from the packaging cells (i.e.from the packaging cells only, or from the packaging cells and from thesupernatant), step e) can be preceded by a step allowing the disruptionof the packaging cell membrane. This step leads to the liberation of theOrthopoxviruses from the packaging cells. The disruption of thepackaging cell membrane can be induced by various techniques well knownby the one skilled in the art. These techniques comprise but are notlimited to freeze/thaw, hypotonic lysis, sonication (by using asonicator) and microfluidization (by using a microfluidizer). Sonicatorsare commercially available from e.g. Heraeus PSP, Biologics, Misonix orGlenMills. Preferred sonicators used according to the present inventionare SONITUBE 20 kHz type SM 20-120-3, SONITUBE 36 kHz type SM 35/3WU andSONITUBE 35 kHz type SM 35-400-3 (Heraeus PSP). Microfluidizers arecommercially available from e.g. Microfluidics Corporation. Thepackaging cell membrane can also be disrupted by using a using a SLMAminco French press. The packaging cell membrane can also be disruptedby using a high speed homogenizer. High speed homogenizers arecommercially available from e.g. Silverson Machines or Ika-Labotechnik.Preferred high speed homogeneizer used according to the presentinvention is a SILVERSON L4R (Silverson Machines). The mixture obtainedafter disruption of the packaging cell membrane can further be incubatedduring at least 1 hour under agitation in order to allow the degradationby the nuclease(s) previously added (in step d)) of the nucleic acids(e.g. DNA) released from the packaging cells. In a specific embodimentof the invention, step e) is therefore preceded by:

-   -   1. a step allowing the disruption of the packaging cell        membrane, preferably by using a high speed homogenizer or by        sonication; and    -   2. a step of incubation of the mixture obtained in step 1)        during at least 1 hour allowing the degradation by the        nuclease(s) added in step d) of the nucleic acids (e.g. DNA)        released from the packaging cells.

According to the invention, the duration of step 2) is preferablycomprised between 1 and 5 hours, and more preferably 2 hours (asdescribed in Example 1).

According to the invention, the nuclease(s) (previously added (in stepd)) is(are) incubated during step 2) at a temperature comprised between22° C. and 28° C., preferably at a temperature comprised between 23° C.and 27° C., more preferably at a temperature comprised between 24° C.and 26° C., and even more preferably at a temperature of 25° C. (asdescribed in Example 1).

Step f) of addition of monovalent salts to the Orthopoxviruses recoveredin step e) allows under suitable conditions:

-   -   to inhibit the nuclease(s) activity; and    -   to avoid the adsorption of the Orthopoxviruses to the anion        exchange adsorbent in step g), i.e. to avoid the adsorption of        more than 10% of Orthopoxviruses to the anion exchange        adsorbent. Therefore nucleic acids (e.g. DNA) only will be        adsorbed to the anion exchange adsorbent in step g).

Monovalent salts include but are not limited to NaCl and KCl. Preferredmonovalent salts used in step f) are NaCl. According to the invention,the concentration of monovalent salts in step f) is in a range of 200 to300 mM, and preferably 250 mM or 300 mM. According to the invention,step f) is performed at a pH comprised between 7.0 and 9.0, preferablybetween 7.5 and 8.5, and more preferably at a pH of 8.0. According tothe invention, step f) of addition of monovalent salts to theOrthopoxviruses recovered in step e) is preferably performed accordingto conditions described in Example 1, wherein NaCl 250 mM, or morepreferably 300 mM, at pH 8.0 is used.

Step g) of contact the mixture obtained in step f) with an anionexchange adsorbent allows under suitable conditions the capture ofnucleic acids (e.g. DNA) contained in said mixture. The Orthopoxvirusesare not captured by the anion exchange adsorbent due to the treatmentperformed in step f).

According to the invention, step g) is performed at a pH comprisedbetween 7.0 and 9.0, preferably between 7.5 and 8.5, and more preferablyat a pH of 8.0 (as described in Example 1).

According to the invention, the duration of step g) is preferablycomprised between 1 and 3 hours, and is more preferably 1 hour (asdescribed in Example 1).

According to the invention, the functional groups of the anion exchangeadsorbent used in step g) are primary, secondary, tertiary andquaternary amino group such as for instance dimethylaminoethyl (DMAE),diethylaminoethyl (DEAE), trimethylaminoethyl (TMAE), triethylaminoethyl(TEAE), the group —R—CH(OH)—CH₂—N+—(CH₃)₃ (also named Q group; seeStreamline® resins, Pharmacia) and other groups such as for instancepolyethyleneimine (PEI) that already have or will have a formal positivecharge within the pH range of 7.0 to 9.0. Preferred functional groups ofthe anion exchange adsorbent used in step g) are selected from the groupconsisting of dimethylaminoethyl (DMAE), diethylaminoethyl (DEAE),trimethylaminoethyl (TMAE) and triethylaminoethyl (TEAE), and are morepreferably trimethylaminoethyl (TMAE).

The anion exchange adsorbent used in step g) can consist in e.g. abeads-formed matrix or a membrane.

According to a preferred embodiment of the invention, the anion exchangeadsorbent used in step g) consists in a beads-formed matrix. Matrix canbe e.g. agarose, hydrophilic polymer, cellulose, dextran or silica.Chains (e.g. dextran chains) are coupled to the matrix. Functionalgroups as previously described are attached to the chains throughchemically stable bonds (e.g. ether bonds). Preferred functional groupsof the beads-formed matrix are trimethylaminoethyl (TMAE). According tothe invention, the beads of the beads-formed matrix have a diameterhigher than the pore size of filters used for the clarification step h).The beads of the beads-formed matrix have therefore preferably adiameter higher than 8 μm, more preferably a diameter comprised between50 μm and 150 μm, more preferably a diameter comprised between 90 μm and120 μm, and even more preferably a diameter of 120 μm. Based on thepresent characteristic of the invention, the Orthopoxviruses (having adiameter of 200-300 nm) will pass through the pore size of filtersduring the clarification step h) (i.e. the Orthopoxviruses will berecovered in the flow through). Anion exchange adsorbents consisting inbeads-formed matrix used according to the invention are preferablyautoclavable. Autoclavable anion exchange adsorbents consisting inbeads-formed matrix have already been described and some of them arecommercially available such as for instance UNOsphere® Q (BioRad),UNOsphere® S (BioRad), STREAMLINE™ Q Sepharose® XL (AmershamBiosciences), STREAMLINE™ SP Sepharose® XL (Amersham Biosciences) orBioSepra® Q hyperZ (Pall Corporation). Preferred autoclavable anionexchange adsorbent consisting in a beads-formed matrix according to thepresent invention is UNOsphere® (BioRad). UNOsphere® Q (BioRad) consistsin hydrophilic spherical polymeric beads having a diameter of 120 μm andearring trimethylaminoethyl (TMAE) functional groups. Step g) of contactthe mixture obtained in step f) with an anion exchange adsorbent whereinsaid exchange adsorbent consists in a beads-formed matrix is preferablyperformed according to the conditions described in Example 1, whereinUNOsphere® Q (BioRad) is used.

According to another preferred embodiment of the invention, the anionexchange adsorbent used in step g) consists in a membrane. Functionalgroups of the membrane can be as previously described. Preferredfunctional groups of the membrane are trimethylaminoethyl (TMAE).According to the present invention, the membrane has a pore size higherthan the diameter of Orthopoxviruses (i.e. 200 nm). The Orthopoxviruseswill be therefore recovered in the flow through. With this regard, themembrane has according to the invention a pore size comprised between 1and 5 μm, and preferably a pore size of 3 μm. Anion exchange adsorbentsconsisting in membranes used according to the invention are preferablyautoclavable. Autoclavable anion exchange adsorbents consisting inmembranes have already been described and some of them are commerciallyavailable such as for instance Sartobind® 75 Q (Sartorius), CUNOPolyNet™ Filters (e.g. PolyNet™ PB P010, P020, P030, P050) or CUNOBetapure™ Filters (e.g. Betapure™ Z13-020, Z13-030, Z13-050). Preferredautoclavable anion exchange adsorbent consisting in a membrane accordingto the present invention is Sartobind® 75 Q (Sartorius).

When step g) is performed with an anion exchange adsorbent being amembrane, the following step h) of clarification (allowing thewithdrawal of the cellular debris) is not required. The cellular debrishave been retained by membranes having a pore size comprised between 1and 5 μm, and preferably a pore size of 3 μm.

Step h) of clarification of the mixture obtained in step g) allows undersuitable conditions the withdrawal of the cellular debris. According tothe invention, the clarification of step h) is preferably performed bydepth filtration. Depth filtration includes but is not limited to theuse of one or more commercially available products such as Sartopure®filters from Sartorius (e.g. Sartopure® PP2), CUNO Incorporated APseries depth filters (e.g. AP01), CUNO Incorporated CP series depthfilters (e.g. CP10, CP30, CP50, CP60, CP70, CP90), CUNO Incorporated HPseries depth filters (e.g. HP10, HP30, HP50, HP60, HP70, HP90), CUNOIncorporated Calif. series depth filters (e.g. CA10, CA30, CA50, CA60,CA70, CA90), CUNO Incorporated SP series depth filters (e.g. SP10, SP30,SP50, SP60, SP70, SP90), CUNO Delipid and Delipid Plus filters,Millipore Corporation CE series depth filters (e.g. CE15, CE20, CE25,CE30, CE35, CE40, CE45, CE50, CE70, CE75), Millipore Corporation DEseries depth filters (e.g. DE25, DE30, DE35, DE40, DE45, DE50, DE55,DE560, DE65, DE70, DE75), Millipore Corporation HC filters (e.g. A1HC,B1HC, COHC), CUNO PolyNet™ Filters (e.g. PolyNet™ PB P050, P100, P200,P300, P400, P500, P700), Millipore Clarigard and Polygard filters, CUNOLife Assure filters, ManCel Associates depth filters (e.g. PR 12 UP,PR12, PR 5 UP); and PALL or SeitzSchenk Incorporated filters. In orderto improve the clarification capacity of the available depth filtrationunits, it can be useful to couple two or more units with decreasing poresizes. In this embodiment, the mixture to be clarified passes throughthe first depth filtration unit where the biggest contaminants areretained and subsequently passes through the second depth filtrationunit. With this regard, according to a preferred embodiment of theinvention, the clarification of step h) is performed by depthfiltration, preferably over filters having a pore size of 8 μm coupledto filters having a pore size of 5 μm. Preferred filters having a poresize of 8 μm and 5 μm used according to the present invention areSartopure® filters commercially available from Sartorius (Sartopure®PP2). The depth filtration may be performed at a flow rate of at least 1L/minute, and preferably at a flow rate of 1 L/minute. According to theinvention, step h) is performed at a pH comprised between 7.0 and 9.0,preferably between 7.5 and 8.5, and more preferably at a pH of 8.0. Steph) of clarification of the mixture obtained in step g) is preferablyperformed according to conditions described in Example 1, wherein theclarification is performed by depth filtration over filters having apore size of 8 μm coupled to filters having a pore size of 5 μm, and ata flow rate of 1 L/minute.

The Orthopoxviruses (having a diameter of 200-300 nm) pass through thepore size of filters during the clarification step h). TheOrthopoxviruses are therefore recovered in the flow through.

Step i) of washing of the anion exchange adsorbent with a solutioncomprising monovalent salts allows under suitable conditions to recoverthe remained Orthopoxviruses in the flow through. Monovalent salts usedinclude but are not limited to NaCl and KCl. Preferred monovalent saltsused in step i) are NaCl. According to the invention, the concentrationof monovalent salts used in step i) is in a range of 200 to 300 mM, andpreferably 250 mM or 300 mM. According to the invention, step i) isperformed at a pH comprised between 7.0 and 9.0, preferably between 7.5and 8.5, and more preferably at a pH of 8.0. According to a preferredembodiment of the invention, the solution comprising monovalent salts instep i) is a pharmaceutically acceptable solution comprising 100 mMTris-HCl, sucrose 5% (w/v), 10 mM sodium glutamate and 50 mM NaCl, pH8.0 with physiological osmolarity (290 mOsm/kg) (i.e. SO8 buffer).

According to other preferred embodiments of the invention, the solutioncomprising monovalent salts in step i) is a pharmaceutically acceptablesolution comprising for instance a Tris buffer, a triethanolamine bufferor a phosphate buffer. Step i) of washing of the anion exchangeadsorbent with a solution comprising monovalent salts is preferablyperformed according to the conditions described in Example 1, whereinthe washing is performed with SO8 pharmaceutically acceptable buffercomprising NaCl 250 mM, or more preferably 300 mM.

Step j) of concentration of the flow through obtained in step h) and theflow through obtained in step i) allows the elimination of the proteinspresent in said flow through fractions.

In a preferred embodiment of the invention, the concentration step j) isperformed by microfiltration. Microfiltration is a pressure drivenmembrane process that concentrates and purifies large molecules. Morespecifically, a solution is passed through filters whose pore size hasbeen chosen to reject the Orthopoxviruses in the retentate and allowsmall molecules (e.g. proteins) to pass through the filters into thepermeate. Microfiltration reduces the volume of the extraction solution.With this regard, the microfiltration is therefore performed by usingfilters having a pore size lower than 0.2 μm, preferably a pore sizecomprised between 0.09 and 0.15 μm, and more preferably a pore size of0.1 μm. Filters used according to the invention are preferablyautoclavable. Autoclavable filters used in step j) are commerciallyavailable such as for instance Prostak Microfiltration Modules(Millipore) wherein Prostak Microfiltration Module PSVVAG021, PSVVAG041and SK2P12E1 are preferred. Step j) of concentration of the flow throughobtained in step h) and the flow through obtained in step i) ispreferably performed according to the conditions described in Example 1,wherein the concentration is performed by microfiltration over filtershaving a pore size of 0.1 μm.

In another preferred embodiment of the invention, the concentration stepj) is performed by ultrafiltration. According to the invention, theultrafiltration is preferably a cross-flow filtration. The principle ofcross-flow filtration is known to the person skilled in the art (see e.g. Richards, G. P. and Goldmintz, D., J. Virol. Methods (1982), 4 (3),pages 147-153. “Evaluation of a cross-flow filtration technique forextraction of polioviruses from inoculated oyster tissue homogenates”).

Step k) of diafiltration of the fraction comprising the Orthopoxvirusesobtained in step j) (e.g. the retentate when the concentration step k)has been performed by microfiltration or by ultrafiltration) is animprovement of microfiltration and involves diluting said fractioncomprising the Orthopoxviruses with a solution to effect a reduction inthe concentration of the impurities in said fraction. The dilution ofthe fraction comprising the Orthopoxviruses allows washing out more ofthe impurities from said fraction. It is understood that thediafiltration may be carried out in a batch mode, semi-continuous mode,or a continuous mode. The diafiltration step k) can be advantageouslyused to change the buffer in which the Orthopoxvirus is comprised. Forexample, it can be useful to exchange the buffer used in thepurification process against a pharmaceutically acceptable buffer.According to the invention, the microfiltration is performed by usingfilters having a pore size lower than 0.2 μm, preferably a pore sizecomprised between 0.09 and 0.15 μm, and more preferably a pore size of0.1 μm. Filters used according to the invention are preferablyautoclavable. Autoclavable filters used in step k) are commerciallyavailable such as for instance Prostak Microfiltration Modules(Millipore) wherein Prostak Microfiltration Module PSVVAG021, PSVVAG041and SK2P12E1 are preferred. Step k) of diafiltration of the fractioncomprising the Orthopoxviruses obtained in step j) is preferablyperformed according to the conditions described in Example 1, whereinthe diafiltration is performed over filters having a pore size of 0.1μm.

Step j) of concentration and step k) of diafiltration can advantageouslybe done with the same type of filters.

The present method A of the invention can further comprise:

-   -   1. a step of gel filtration (i.e. step l)); and    -   2. a step of diafiltration (i.e. step m)).

Gel filtration step (i.e. step l): According to the invention, thesample obtained in step k) is treated on a solid support comprisingbeads having a diameter comprised between 3 and 160 μm, advantageouslybetween 80 and 160 μm, preferably between 40 and 105 μm, more preferablybetween 25 and 75 μm, more preferably between 20 and 80 μm, and evenmore preferably between 20 and 60 μm. According to the invention, saidsupport has a porosity closed to the diameter of the Orthopoxvirus (i.e.200-300 nm) so that the latter does not penetrate into the beads. On theother hand, the molecules which are smaller in size penetrate into thebeads and the migration thereof is slowed. The supports used in step l)of gel filtration can be based e.g. on agarose, dextran, acrylamide,silica, ethylene glycol/methacrylate copolymers, or mixtures thereofsuch as for instance mixtures of agarose and dextran. According to theinvention, the supports are preferably used without functional groups.Gel filtration chromatography supports are commercially available suchas for instance:

-   -   Ethylene glycol/methacrylate gel filtration chromatography        supports (e.g. Toyopearl® HW 55, Toyopearl® HW 65 and Toyopearl®        HW 75, having a bead diameter comprised between 20 and 60 μm,        Tosohaas);    -   Allyl dextran/methylene bisacrylamide gel filtration        chromatography supports (e.g. Sephacryl™ S300 HR having a bead        diameter comprised between 25 and 75 μm; Sephacryl™ S400 HR        having a bead diameter comprised between 25 and 75 μm;        Sephacryl™ S500 HR having a bead diameter comprised between 25        and 75 μm; Sephacryl™ S1000 SF having a bead diameter comprised        between 40 and 105 μm, all from Pharmacia);    -   N-acrylaminohydroxypropanediol gel filtration chromatography        supports (e.g. Trisacryl having a bead diameter comprised        between 80 and 160 μm, Biosepra);    -   Agarose gel filtration chromatography supports (e.g. Macro-Prep        SE having a bead diameter comprised between 20 and 80 μm,        Bio-Rad).

Ethylene glycol/methacrylate gel filtration chromatography supports(e.g. Toyopearl® HW 55, Toyopearl® HW 65 and Toyopearl® HW 75, having abead diameter comprised between 20 and 60 μm, Tosohaas) are preferred.

Preferred conditions for step l) of gel filtration according to theinvention are:

-   -   a concentration of monovalent salts selected from NaCl and KCl,        preferably NaCl, in a range of 200 mM to 2 M, preferably in a        range of 200 mM to 1 M, and more preferably at 500 mM;    -   a pH comprised between 7.0 and 9.0, preferably between 7.5 and        8.5, and more preferably a pH of 8.0.

The step m) of diafiltration is performed by means and conditions aspreviously described in step k) of diafiltration.

According to the present invention, each step from step f) to step l) ofMethod A previously described can be preceded by a step of incubating ofthe sample comprising the Orthopoxviruses in presence of one or morestabilizers. As used herein, “stabilizers” refers to agents allowing thepreservation of the Orthopoxviruses. Stabilizers include but are notlimited to saccharides (e.g. sucrose, trehalose, sorbose, melezitose,sorbitol, stachyose, raffinose, fructose, mannose, maltose, lactose,arabinose, xylose, ribose, rhamnose, galactose, glucose, mannitol,xylitol, erythritol, threitol, sorbitol, glycerol), amino acids (e.g.Gly; Leu; Lys; Arg; Asp; Val; Glu), detergents (e.g. Triton X-100; Tweensuch as for instance Tween 20, Tween 80 or Tween 85) and salts (e.g.NaCl; KCl). According to a preferred embodiment of the invention, thestabilizers used in this step of incubation are saccharides and morepreferably saccharose. According to the invention, the concentration ofsaccharose used in this step of incubation is in a range of 25 to 100g/L, and preferably is 50 g/L. According to the invention, thestabilizer(s) can be comprised in a solution consisting of apharmaceutically acceptable solution comprising 100 mM Tris-HCl, sucrose5% (w/v), 10 mM sodium glutamate and 50 mM NaCl, pH 8.0 withphysiological osmolarity (290 mOsm/kg) (i.e. SO8 buffer). According tothe invention, the stabilizer(s) can be comprised in a solutionconsisting of a pharmaceutically acceptable solution comprising forinstance a Tris buffer, a triethanolamine buffer or a phosphate buffer.According to the invention, this step of incubation in presence ofstabilizers is performed at a pH comprised between 7.0 and 9.0,preferably between 7.5 and 8.5, and more preferably at a pH of 8.0.According to the invention, the duration of this step of incubation inpresence of stabilizers is comprised between 1 hour and 20 hours, andmore preferably 18 hours. According to the invention, this step ofincubation in presence of stabilizers is performed at a temperaturecomprised between 2 and 8° C., preferably at a temperature comprisedbetween 3° C. and 7° C., more preferably at a temperature comprisedbetween 4° C. and 6° C., and even more preferably at a temperature of 5°C. This step of incubation in presence of stabilizers is preferablyperformed in presence of 50 g/L saccharose during 18 hours at atemperature of 5° C. as described in Example 1.

According to another embodiment of the Method A of the invention aspreviously described, the clarification step (step h)) is performedafter the step e) of recovering the Orthopoxviruses from the culturesupernantant and/or the packaging cells.

According to another embodiment of the invention, step f) of addition ofmonovalent salts to the Orthopoxviruses recovered in step e) undersuitable conditions to inhibit the nuclease(s) activity and to avoid theadsorption of said Orthopoxviruses to the anion exchange adsorbent instep g), is not performed.

With this regard, the present invention therefore also relates to amethod (i.e. Method B) for producing and purifying a wild type, anattenuated and/or a recombinant Orthopoxvirus, comprising the followingsteps:

-   -   a′) preparing a culture of packaging cells;    -   b′) infecting the packaging cell culture with an Orthopoxvirus;    -   c′) culturing the infected packaging cells until progeny        Orthopoxvirus is produced;    -   d′) incubation in presence of one or more nucleases;    -   e′) recovering the Orthopoxviruses from the culture supernatant        and/or the packaging cells;    -   f′) incubating the Orthopoxviruses recovered in step e′) in        presence of:        -   1. one or more agents capable to inhibit the nuclease(s)            activity, and optionally        -   2. one or more stabilizers.    -   g′) contacting the mixture obtained in step f′) with an anion        exchange adsorbent under suitable conditions to allow the        capture of the Orthopoxviruses and nucleic acids;    -   h′) clarifying the mixture obtained in step g′) under suitable        conditions to allow the withdrawal of the cellular debris;    -   l′) eluting the Orthopoxviruses with a solution comprising        monovalent salts;    -   j′) concentrating the mixture obtained in step i′);    -   k′) diafiltrating the fraction comprising the Orthopoxviruses        obtained in step j′).

Step a′) of preparation of a culture of packaging cells refers to stepa) of preparation of a culture of packaging cells as previouslydescribed.

Step b′) of infection of the packaging cell culture with anOrthopoxvirus refers to step b) of infection of the packaging cellculture with an Orthopoxvirus as previously described.

Step c′) of culture of the infected packaging cells until progenyOrthopoxvirus is produced refers to step c) of culture of the infectedcells until progeny Orthopoxvirus is produced as previously described.

Step d′) of incubation in presence of one or more nucleases refers tostep d) of incubation in presence of one or more nucleases as previouslydescribed.

Step e′) of recovering of the Orthopoxviruses from the culturesupernatant and/or the packaging cells refers to step e) of recoveringof the Orthopoxviruses from the culture supernatant and/or the packagingcells as previously described.

The Orthopoxviruses recovered in step e′) are then incubated (step f′))in presence of:

-   -   1. one or more agents capable to inhibit the nuclease(s)        activity, and optionally    -   2. one or more stabilizers.

Agents capable to inhibit the nuclease activity include but are notlimited to chelating agents (e.g. ethylenediamine tetraacetate (EDTA);diethylenetriamine pentaacetate

(DTPA); nitrilotriacetate (NTA)), monovalent salts (e.g. NaCl or KCl),proteases, phosphate, monovalent cations (e.g. Na⁺; Li⁺; K⁺; Ag⁺),guanidine hydrochloride and ammonium sulfate. Chelating agents such ase.g. EDTA, DTPA or NTA, remove metal ions (e.g. Mg²⁺; Mn²⁺) whichconstitute, as previoulsy described, essential cofactors for theactivity of nuclease(s). Monovalent salts (e.g. NaCl or KCl) lead to theinactivation of nucleases at a concentration comprised between 50 and150 mM, and preferably at about 100 mM. Phosphate and/or monovalentcations (e.g. Na⁺; Li⁺; K⁺; Ag⁺) lead to the inactivation of nucleasesat a concentration of about or above 100 mM. Guanidine hydrochloride andammonium sulfate lead to the inactivation of nucleases at aconcentration above 100 mM. According to a preferred embodiment of theinvention, the agents capable to inhibit the nuclease activity in stepf′) are chelating agents, and more preferably ethylenediaminetetraacetate (EDTA). According to the invention, the concentration ofEDTA used in step f′) is in a range of 5 to 20 mM, and preferably is 10mM. According to another preferred embodiment of the invention, theagents capable to inhibit the nuclease activity in step f′) aremonovalent salts, preferably NaCl, and more preferably NaCl 100 mM.According to another preferred embodiment of the invention, the agentscapable to inhibit the nuclease activity in step f′) are monovalentsalts and chelating agents, preferably NaCl and EDTA, and morepreferably NaCl 100 mM and EDTA 10 mM (as described in Example 4).

According to the invention, step f′) is performed at a pH comprisedbetween 7.0 and 9.0, preferably between 7.5 and 8.5, and more preferablyat a pH of 8.0 (as described in Example 4).

According to the invention, step f′) is performed at a temperaturecomprised between 2° C. and 8° C., preferably at a temperature comprisedbetween 3° C. and 7° C., more preferably at a temperature comprisedbetween 4° C. and 6° C., and even more preferably at a temperature of 5°C. According to the invention, the duration of step f′) is comprisedbetween 5 minutes and 20 hours. In a preferred embodiment of theinvention, the duration of step f′) is comprised between 2 and 20 hours,and is preferably 18 hours. In this present embodiment, theOrthopoxviruses obtained in step e′) are also incubated in presence ofone or more stabilizers, in addition to agent(s) capable to inhibit thenuclease(s) activity (as previously described). “Stabilizers” refers instep f′) to agents allowing the preservation of the Orthopoxvirusesduring the treatment with agent(s) capable to inhibit the nuclease(s)activity in said step f′). Stabilizers include but are not limited tosaccharides (e.g. sucrose, trehalose, sorbose, melezitose, sorbitol,stachyose, raffinose, fructose, mannose, maltose, lactose, arabinose,xylose, ribose, rhamnose, galactose, glucose, mannitol, xylitol,erythritol, threitol, sorbitol, glycerol), amino acids (e.g. Gly; Leu;Lys; Arg; Asp; Val; Glu), detergents (e.g. Triton X-100; Tween such asfor instance Tween 20, Tween 80 or Tween 85) and salts (e.g. NaCl; KCl).According to a preferred embodiment of the invention, the stabilizersused in step f′) are saccharides, and preferably saccharose. Accordingto the invention, the concentration of saccharose used in step f′) is ina range of 25 to 100 g/L, and preferably is 50 g/L. According to theinvention, the stabilizer(s) can be comprised in a solution consistingof a pharmaceutically acceptable solution comprising 100 mM Tris-HCl,sucrose 5% (w/v), 10 mM sodium glutamate and 50 mM NaCl, pH 8.0 withphysiological osmolarity (290 mOsm/kg) (i.e. SO8 buffer). According tothe invention, the stabilizer(s) can be comprised in a solutionconsisting of a pharmaceutically acceptable solution comprising forinstance a Tris buffer, a triethanolamine buffer or a phosphate buffer.

Step g′) of contact of the mixture obtained in step f′) with an anionexchange adsorbent allows under suitable conditions the capture of saidOrthopoxvirus and nucleic acids (e.g. DNA) as well contained in saidmixture.

According to the invention, step g′) is performed at a pH comprisedbetween 7.0 and 9.0, preferably between 7.5 and 8.5, and more preferablyat a pH of 8.0 (as described in Example 4).

According to the invention, the duration of step g′) is preferablycomprised between 1 and 3 hours, and is more preferably 1 hour (asdescribed in Example 4).

According to the invention, the functional groups of the anion exchangeadsorbent used in step g′) are primary, secondary, tertiary andquaternary amino group such as for instance dimethylaminoethyl (DMAE),diethylaminoethyl (DEAE), trimethylaminoethyl (TMAE), triethylaminoethyl(TEAE), the group —R—CH(OH)—CH₂—N+—(CH₃)₃ (also named Q group; seeStreamline® resins, Pharmacia) and other groups such as for instancepolyethyleneimine (PEI) that already have or will have a formal positivecharge within the pH range of 7.0 to 9.0. Preferred functional groups ofthe anion exchange adsorbent used in step g′) are selected from thegroup consisting of dimethylaminoethyl (DMAE), diethylaminoethyl (DEAE),trimethylaminoethyl (TMAE) and triethylaminoethyl (TEAE), and are morepreferably trimethylaminoethyl (TMAE).

The anion exchange adsorbent used in step g′) can consist in e.g. abeads-formed matrix or a membrane.

According to a preferred embodiment of the invention, the anion exchangeadsorbent used in step g′) consists in a beads-formed matrix. Matrix canbe e.g. agarose, hydrophilic polymer, cellulose, dextran or silica.Chains (e.g. dextran chains) are coupled to the matrix. Functionalgroups as previously described are attached to the chains throughchemically stable bonds (e.g. ether bonds). Preferred functional groupsof the beads-formed matrix are trimethylaminoethyl (TMAE). According tothe invention, the beads of the beads-formed matrix have a diameterhigher than the pore size of filters used for the clarification steph′). The beads of the beads-formed matrix have therefore preferably adiameter higher than 8 μm, more preferably a diameter comprised between50 μm and 150 μm, more preferably a diameter comprised between 90 μm and120 μm, and even more preferably a diameter of 120 μm. Based on thepresent characteristic of the invention, the beads of the beads-formedmatrix caring the Orthopoxviruses will be retained by the filters duringthe clarification step h′) as well as the cellular debris. Anionexchange adsorbents consisting in beads-formed matrix used according tothe invention are preferably autoclavable. Autoclavable anion exchangeadsorbents consisting in beads-formed matrix have already been describedand some of them are commercially available such as for instanceUNOsphere® 0 (BioRad), UNOsphere® S (BioRad), STREAMLINE™ Q Sepharose®XL (Amersham Biosciences), STREAMLINE™ SP Sepharose® XL (AmershamBiosciences) or BioSepra® hyperZ (Pall Corporation). Preferredautoclavable anion exchange adsorbent consisting in a beads-formedmatrix according to the present invention is UNOsphere® (BioRad) andBioSepra® Q hyperZ (Pall Corporation). UNOsphere® Q (BioRad) consists inhydrophilic spherical polymeric beads having a diameter of 120 μm andcarring trimethylaminoethyl (TMAE) functional groups. BioSepra® Q hyperZ(Pall Corporation) consists in highly dense and porous zirconium dioxidebeads having a diameter of about 75 μm and carring quaternary amine Qfunctional groups. Step g′) of contact the mixture obtained in step f′)with an anion exchange adsorbent wherein said exchange adsorbentconsists in a beads-formed matrix is preferably performed according tothe conditions described in Example 4, wherein BioSepra® Q hyperZ (PallCorporation) is used.

According to another preferred embodiment of the invention, the anionexchange adsorbent used in step g′) consists in a membrane. Functionalgroups of the membrane can be as previously described. Preferredfunctional groups of the membrane are trimethylaminoethyl (TMAE).According to a preferred embodiment of the invention, the membrane usedin step g′) has a pore size comprised between 1 and 5 μm, and preferablya pore size of 3 μm. According to another preferred embodiment of theinvention, the membrane used in step g′) has a pore size lower than thesize of Orthopoxviruses (i.e. 200 nm), and more particularly a pore sizeof 0.1 μm. Many anion exchange adsorbents consisting in membranes havealready been described and some of them are commercially available suchas for instance Sartobind® 75 Q (Sartorius). Anion exchange adsorbentsconsisting in membranes used according to the invention are preferablyautoclavable. Autoclavable anion exchange adsorbents consisting inmembranes have already been described and some of them are commerciallyavailable such as for instance Sartobind® 75 Q (Sartorius). Preferredautoclavable anion exchange adsorbent consisting in a membrane accordingto the present invention is Sartobind® 75 Q (Sartorius).

When step g′) is performed with an anion exchange adsorbent being ananion exchange membrane, the following step h′) of clarification(allowing the withdrawal of the cellular debris) is not required. Thecellular debris have been retained by anion exchange membranes (aspreviously described) used in step f′).

Step h′) of clarification of the mixture obtained in step g′) refers tostep h) of clarification of the mixture obtained in step g) aspreviously described.

According to a preferred embodiment of the invention, when step g′) isperformed with an anion exchange adsorbent being a beads-formed matrix,step h′) can be preceded by a step allowing the removal of saidbeads-formed matrix caring the Orthopoxviruses. With this regard, saidstep is performed by using e.g. filter bags having therefore a pore sizelower than the size of the beads beads-formed matrix. According to theinvention, the pore size of the filter bags is comprised between 10 and100 μm, preferably between 25 and 100 μm, and more preferably 50 μm.Filter bags used according to the invention are preferably autoclavable.Autoclavable filter bags have already been described and some of themare commercially available such as for instance CUNO™ Felt Filter Bags(CUNO) wherein polyester Felt Filter Bags (e.g. NB EES 0010, 0025, 0050,0100), polyester/polypropylene Felt Filter Bags (e.g. NB PES 0010, 0025,0050, 0100) and nylon monofilament Felt Filter Bags (e.g. NB NYS 0025,0050, 0100) are preferred.

Step i′) of elution of the Orthopoxviruses with a solution comprisingmonovalent salts allows the recovering of the captured Orthopoxvirusesfrom the anion exchange adsorbent. Monovalent salts used include but arenot limited to NaCl and KCl. Preferred monovalent salts used in step i′)are NaCl. According to one embodiment of the invention, the elution isperformed by an increasing monovalent salts concentration gradient,ranging preferably from of 0 to 2.5 M, more preferably from of 0 to 2 Mand even more preferably from of 0 to 1.5 M. According to anotherembodiment of the invention, the elution is performed by a single-stepelution at a concentration of monovalent salts which is below 1M,preferably comprised between 300 mM and 750 mM, and more preferably of500 mM. According to the invention, step i′) is performed at a pHcomprised between 7.0 and 9.0, preferably between 7.5 and 8.5, and morepreferably at a pH of 8.0. According to a preferred embodiment of theinvention, the solution comprising monovalent salts in step i′) is apharmaceutically acceptable solution comprising 100 mM Tris-HCl, sucrose5% (w/v), 10 mM sodium glutamate and 50 mM NaCl, pH 8.0 withphysiological osmolarity (290 mOsm/kg) (i.e. SO8 buffer). According toother preferred embodiments of the invention, the solution comprisingmonovalent salts in step i′) is a pharmaceutically acceptable solutioncomprising for instance a Tris buffer, a triethanolamine buffer or aphosphate buffer. Step i′) of elution of the Orthopoxviruses with asolution comprising monovalent salts is preferably performed accordingto the conditions described in Example 4, wherein the elution isperformed by an increasing NaCl concentration gradient (300 mM; 400 mM;500 mM) in SO8 buffer.

Step j′) of concentration of the mixture obtained in step i′) allows theelimination of the proteins present in said flow through fractions.

In a preferred embodiment of the invention, the concentration step j′)is performed by microfiltration (as previously described in step j)).

In another preferred embodiment of the invention, the concentration stepj′) is performed by ultrafiltration (as previously described in stepj)).

Step k′) of diafiltration of the fraction comprising the Orthopoxvirusesobtained in step j′) refers to step k) of diafiltrating the fractioncomprising the Orthopoxviruses obtained in step j) as previouslydescribed.

The present method B of the invention can further comprise:

-   -   1. a step of gel filtration (i.e. step l′)); and    -   2. a step of diafiltration (i.e. step m′)).

Step l′) of gel filtration refers to step l) of gel filtration.

Step m′) of diafiltration refers to step m) of diafiltration.

According to the present invention, each step from step f′) to step l′)of the Method B previously described may be preceded by a step ofincubating of the sample comprising the Orthopoxviruses in presence ofone or more stabilizers. As used herein, “stabilizers” refers to agentsallowing the preservation of the Orthopoxviruses. Stabilizers includebut are not limited to saccharides (e.g. sucrose, trehalose, sorbose,melezitose, sorbitol, stachyose, raffinose, fructose, mannose, maltose,lactose, arabinose, xylose, ribose, rhamnose, galactose, glucose,mannitol, xylitol, erythritol, threitol, sorbitol, glycerol), aminoacids (e.g. Gly; Leu; Lys; Arg; Asp; Val; Glu), detergents (e.g. TritonX-100; Tween such as for instance Tween 20, Tween 80 or Tween 85) andsalts (e.g. NaCl; KCl). According to a preferred embodiment of theinvention, the stabilizers used in this step of incubation aresaccharides and more preferably saccharose. According to the invention,the concentration of saccharose used in this step of incubation is in arange of 25 to 100 g/L, and preferably is 50 g/L. According to theinvention, the stabilizer(s) can be comprised in a solution consistingof a pharmaceutically acceptable solution comprising 100 mM Tris-HCl,sucrose 5% (w/v), 10 mM sodium glutamate and 50 mM NaCl, pH 8.0 withphysiological osmolarity (290 mOsm/kg) (i.e. SO8 buffer). According tothe invention, the stabilizer(s) can be comprised in a solutionconsisting of a pharmaceutically acceptable solution comprising forinstance a Tris buffer, a triethanolamine buffer or a phosphate buffer.According to the invention, this step of incubation in presence ofstabilizers is performed at a pH comprised between 7.0 and 9.0,preferably between 7.5 and 8.5, and more preferably at a pH of 8.0.According to the invention, the duration of this step of incubation inpresence of stabilizers is comprised between 1 hour and 20 hours, andmore preferably 18 hours. According to the invention, this step ofincubation in presence of stabilizers is performed at a temperaturecomprised between 2° C. and 8° C., preferably at a temperature comprisedbetween 3° C. and 7° C., more preferably at a temperature comprisedbetween 4° C. and 6° C., and even more preferably at a temperature of 5°C. This step of incubation in presence of stabilizers is preferablyperformed in presence of 50 g/L saccharose during 18 hours at atemperature of 5° C.

According to another embodiment of the Method B of the invention aspreviously described, the clarification step (step h′)) is performedafter the step e′) of recovering the Orthopoxviruses from the culturesupernantant and/or the packaging cells.

The present invention also related to methods that combine step(s) ofMethod A and step(s) of Method B, as previously described.

With this regard, the present invention more particularly relates to amethod (i.e. Method C) for producing and purifying a wild type, anattenuated and/or a recombinant Orthopoxvirus, comprising the followingsteps:

-   -   a″) preparing a culture of packaging cells;    -   b″) infecting the packaging cell culture with an Orthopoxvirus;    -   c″) culturing the infected packaging cells until progeny        Orthopoxvirus is produced;    -   d″) incubation in presence of one or more nucleases;    -   e″) recovering the Orthopoxviruses from the culture supernatant        and/or the packaging cells;    -   f″) incubating the Orthopoxviruses recovered in step e″) in        presence of:        -   1. one or more agents capable to inhibit the nuclease(s)            activity, and optionally        -   2. one or more stabilizers;    -   g″) contacting the mixture obtained in step f″) with an anion        exchange adsorbent under suitable conditions to allow the        capture of said Orthopoxviruses and nucleic acids;    -   h″) clarifying the mixture obtained in step g″) under suitable        conditions to allow the withdrawal of the cellular debris;    -   i″) eluting the Orthopoxviruses with a solution comprising        monovalent salts;    -   j″) adding monovalent salts to the Orthopoxviruses eluted in        step i″) in order to avoid the adsorption of said        Orthopoxviruses to the anion exchange adsorbent in step k″);    -   k″) contacting the mixture obtained in step j″) with an anion        exchange adsorbent under suitable conditions to allow the        capture of nucleic acids;    -   l″) washing of the anion exchange adsorbent with a solution        comprising monovalent salts under suitable conditions to recover        the remained Orthopoxviruses in the flow through;    -   m″) concentrating the flow through obtained in step l″);    -   n″) diafiltrating the fraction comprising the Orthopoxviruses        obtained in step m″).

Step a″) of preparation of a culture of packaging cells refers to stepa) of preparation of a culture of packaging cells as previouslydescribed.

Step b″) of infection of the packaging cell culture with anOrthopoxvirus refers to step b) of infection of the packaging cellculture with an Orthopoxvirus as previously described.

Step c″) of culture of the infected packaging cells until progenyOrthopoxvirus is produced refers to step c) of culture of the infectedcells until progeny Orthopoxvirus is produced as previously described.

Step d″) of incubation in presence of one or more nucleases refers tostep d) of incubation in presence of one or more nucleases as previouslydescribed.

Step e″) of recovering of the Orthopoxviruses from the culturesupernatant and/or the packaging cells refers to step e) of recoveringof the Orthopoxviruses from the culture supernatant and/or the packagingcells as previously described.

Step f″) of incubation of the Orthopoxviruses recovered in step e″) inpresence of (1) one or more agents capable to inhibit the nuclease(s)activity, and optionally (2) one or more stabilizers, refers to step f′)of incubation of the Orthopoxviruses recovered in step e′) in presenceof (1) one or more agents capable to inhibit the nuclease(s) activity,and optionally (2) one or more stabilizers, as previously described.

Step g″) of contact the mixture obtained in step f″) with an anionexchange adsorbent under suitable conditions to allow the capture ofsaid Orthopoxviruses and nucleic acids, refers to step g′) of contactthe mixture obtained in step f′) with an anion exchange adsorbent undersuitable conditions to allow the capture of said Orthopoxviruses andnucleic acids, as previously described.

Step h″) of clarification of the mixture obtained in step g″) refers tostep h) of clarification of the mixture obtained in step g) aspreviously described.

Step i″) of elution of the Orthopoxviruses with a solution comprisingmonovalent salts refers to step i′) of elution of the Orthopoxviruseswith a solution comprising monovalent salts as previously described.

Step j″) of addition of monovalent salts to the Orthopoxvirusespreviously eluted in step i″) allows under suitable conconditions toavoid the adsorption of said Orthopoxviruses to the anion exchangeadsorbent in step k″) (i.e. to avoid the adsorption of more than 10% ofOrthopoxviruses to the anion exchange adsorbent). Therefore nucleicacids (e.g. DNA) only will be adsorbed to the anion exchange adsorbentin step k″). Monovalent salts include but are not limited to NaCl andKCl. Preferred monovalent salts used in step j″) are NaCl. According tothe invention, the concentration of monovalent salts in step j″) is in arange of 200 to 300 mM, and preferably 250 mM or 300 mM. According tothe invention, step j″) is performed at a pH comprised between 7.0 and9.0, preferably between 7.5 and 8.5, and more preferably at a pH of 8.0.

Step k″) of contact of the mixture obtained in step j″) with an anionexchange adsorbent under suitable conditions to allow the capture ofnucleic acids, refers to step g) of contact of the mixture obtained instep f) with an anion exchange adsorbent under suitable conditions toallow the capture of nucleic acids as previously described.

Step l″) of washing of the anion exchange adsorbent with a solutioncomprising monovalent salts under suitable conditions to recover theremained Orthopoxviruses in the flow through, refers to step i) ofwashing of the anion exchange adsorbent with a solution comprisingmonovalent salts under suitable conditions to recover the remainedOrthopoxviruses in the flow through as previously described.

Step m″) of concentration of the flow through obtained in step l″)allows the elimination of the proteins present in said flow throughfraction. In a preferred embodiment of the invention, the concentrationstep m″) is performed by microfiltration (as previously described instep j)). In another preferred embodiment of the invention, theconcentration step m″) is performed by ultrafiltration (as previouslydescribed in step j)).

Step n″) of diafiltration of the fraction comprising the Orthopoxvirusesobtained in step m″) refers to step k) of diafiltrating the fractioncomprising the Orthopoxviruses obtained in step j) as previouslydescribed.

The present method C of the invention can further comprise:

-   -   1. a step of gel filtration (i.e. step o″)); and    -   2. a step of diafiltration (i.e. step p″)).

Step o″) of gel filtration refers to step l) of gel filtration.

Step p″) of diafiltration refers to step m) of diafiltration.

According to the present invention, each step from step f″) to step p″)(and preferably each step from step k″) to step p″)) of the Method Cpreviously described may be preceded by a step of incubating of thesample comprising the Orthopoxviruses in presence of one or morestabilizers. As used herein, “stabilizers” refers to agents allowing thepreservation of the Orthopoxviruses. Stabilizers include but are notlimited to saccharides (e.g. sucrose, trehalose, sorbose, melezitose,sorbitol, stachyose, raffinose, fructose, mannose, maltose, lactose,arabinose, xylose, ribose, rhamnose, galactose, glucose, mannitol,xylitol, erythritol, threitol, sorbitol, glycerol), amino acids (e.g.Gly; Leu; Lys; Arg; Asp; Val; Glu), detergents (e.g. Triton X-100; Tweensuch as for instance Tween 20, Tween 80 or Tween 85) and salts (e.g.NaCl; KCl). According to a preferred embodiment of the invention, thestabilizers used in this step of incubation are saccharides and morepreferably saccharose. According to the invention, the concentration ofsaccharose used in this step of incubation is in a range of 25 to 100g/L, and preferably is 50 g/L. According to the invention, thestabilizer(s) can be comprised in a solution consisting of apharmaceutically acceptable solution comprising 100 mM Tris-HCl, sucrose5% (w/v), 10 mM sodium glutamate and 50 mM NaCl, pH 8.0 withphysiological osmolarity (290 mOsm/kg) (i.e. SO8 buffer). According tothe invention, the stabilizer(s) can be comprised in a solutionconsisting of a pharmaceutically acceptable solution comprising forinstance a Tris buffer, a triethanolamine buffer or a phosphate buffer.According to the invention, this step of incubation in presence ofstabilizers is performed at a pH comprised between 7.0 and 9.0,preferably between 7.5 and 8.5, and more preferably at a pH of 8.0.According to the invention, the duration of this step of incubation inpresence of stabilizers is comprised between 1 hour and 20 hours, andmore preferably 18 hours. According to the invention, this step ofincubation in presence of stabilizers is performed at a temperaturecomprised between 2° C. and 8° C., preferably at a temperature comprisedbetween 3° C. and 7° C., more preferably at a temperature comprisedbetween 4° C. and 6° C., and even more preferably at a temperature of 5°C. This step of incubation in presence of stabilizers is preferablyperformed in presence of 50 g/L saccharose during 18 hours at atemperature of 5° C.

According to another embodiment of the Method C of the invention aspreviously described, the clarification step (step h″)) is performedafter the step e″) of recovering the Orthopoxviruses from the culturesupernantant and/or the packaging cells.

According to the present invention, the samples comprising theOrthopoxviruses obtained after each step of the methods of the presentinvention may be preserved by freezing well known from those skilled inthe art.

According to the invention, the methods of the present invention areperformed at a pH comprised between 7.0 and 9.0, preferably between 7.5and 8.5, and more preferably at a pH of 8.0.

In a preferred embodiment of the invention, the Orthopoxvirus is aVaccinia Virus (VV). Preferred VV according to the invention are VV asdescribed for instance in patent applications PCT/EP2008/009720(WO2009/065546 describing VV comprising defective I4L and/or F4Lgene(s)) or PCT/EP2008/009721 (WO2009/065547 describing VV comprisingdefective F2L gene).

In another preferred embodiment of the invention, the Orthopoxvirus is amodified Vaccinia Virus Ankara (MVA). Preferred MVA according to thepresent invention are MVA as deposited before Collection Nationale deCultures de Microorganismes (CNCM) under depositary N° I-721, MVA 575(ECACC V00120707) and MVA-BN (ECACC V00083008).

The term “recombinant Orthopoxvirus” refers to an Orthopoxviruscomprising an exogenous sequence inserted in its genome. As used herein,an exogenous sequence refers to a nucleic acid which is not naturallypresent in the parent Orthopoxvirus.

In one embodiment, the exogenous sequence encodes a molecule having adirectly or indirectly cytotoxic function. By “directly or indirectly”cytotoxic, we mean that the molecule encoded by the exogenous sequencemay itself be toxic (for example ricin, tumour necrosis factor (TNF),interleukin-2 (IL2), interferon-gamma (IFNγ), ribonuclease,deoxyribonuclease, Pseudomonas exotoxin A) or it may be metabolised toform a toxic product, or it may act on something else to form a toxicproduct. The sequence of ricin cDNA is disclosed in Lamb et al (Eur. J.Biochem., 1985, 148, 265-270).

In a preferred embodiment of the invention, the exogenous sequence is asuicide gene. A suicide gene encodes a protein able to convert arelatively non-toxic prodrug to a toxic drug. For example, the enzymecytosine deaminase converts 5-fluorocytosine (5-FC) to 5-fluorouracil(5-FU) (Mullen et al (1922) PNAS 89, 33); the herpes simplex enzymethymidine kinase sensitises cells to treatment with the antiviral agentganciclovir (GCV) or aciclovir (Moolten (1986) Cancer Res. 46, 5276;Ezzedine et al (1991) New Biol 3, 608). The cytosine deaminase of anyorganism, for example E. coli or Saccharomyces cerevisiae, may be used.Thus, in preferred embodiment of the invention, the suicide gene encodesa protein having a cytosine deaminase activity, and more preferably FCU1protein or FCU1-8 protein covered by patent applications WO 99/54481, WO05/07857, PCT/EP2008/009720 and PCT/EP2008/009721 incorporated herein byreference.

With this regard, preferred recombinant Orthopoxviruses producedaccording to the method of the invention are:

-   -   MVA-FCU1 (see WO 99/54481) also called TG4023;    -   MVA-FCU1-8 (see WO 05/07857); and    -   VV-FCU1 wherein said VV comprises more particularly a defective        I4L and/or F4L gene, and a defective J2R gene (see        PCT/EP2008/009720/WO2009/065546 and        PCT/EP2008/009721/WO2009/065547).

Other examples of pro-drug/enzyme combinations include those disclosedby Bagshawe et al (WO 88/07378), namely various alkylating agents andthe Pseudomonas spp. CPG2 enzyme, and those disclosed by Epenetos &Rowlinson-Busza (WO 91/11201), namely cyanogenic pro-drugs (for exampleamygdalin) and plant-derived beta-glucosidases. Enzymes that are usefulin this embodiment of the invention include, but are not limited to,alkaline phosphatase useful for converting phosphate-containing prodrugsinto free drugs; arylsulfatase useful for converting sulfate-containingprodrugs into free drugs; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as beta-galactosidase andneuraminidase useful for converting glycosylated prodrugs into freedrugs; beta-lactamase useful for converting drugs derivatized withbeta-lactams into free drugs; and penicillin amidases, such aspenicillin V amidase or penicillin G amidase, useful for convertingdrugs derivatized at their amine nitrogens with phenoxyacetyl orphenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as abzymes,can be used to convert the prodrugs of the invention into free activedrugs (Massey R. et al., Nature, 1987, 328, 457-458). Similarly,prodrugs include, but are not limited to, the above-listed prodrugs,e.g., phosphate-containing prodrugs, thiophosphate-containing prodrugs,sulfate-containing prodrugs, peptide-containing prodrugs, D-aminoacid-modified prodrugs, glycosylated prodrugs, beta-lactam-containingprodrugs, optionally substituted phenoxyacetamide-containing prodrugs oroptionally substituted phenylacetamide-containing prodrugs,5-fluorocytosine and other 5-fluorouridine prodrugs which can beconverted. Examples of cytotoxic drugs that can be derivatized into aprodrug form for use in this invention include, but are not limited to,etoposide, teniposide, adriamycin, daunomycin, carminomycin,aminopterin, dactinomycin, mitomycins, cis-platinum and cis-platinumanalogues, bleomycins, esperamicins (see for example U.S. Pat. No.4,675,187), 5-fluorouracil, melphalan and other related nitrogenmustards.

In a further embodiment the exogenous gene encodes a ribozyme capable ofcleaving targeted RNA or DNA. The targeted RNA or DNA to be cleaved maybe RNA or DNA which is essential to the function of the cell andcleavage thereof results in cell death or the RNA or DNA to be cleavedmay be RNA or DNA which encodes an undesirable protein, for example anoncogene product, and cleavage of this RNA or DNA may prevent the cellfrom becoming cancerous.

In a still further embodiment the exogenous gene encodes an antisenseRNA. By “antisense RNA” we mean an RNA molecule which hybridises to, andinterferes with the expression from an mRNA molecule encoding a proteinor to another RNA molecule within the cell such as pre-mRNA or tRNA orrRNA, or hybridises to, and interferes with the expression from a gene.

In another embodiment of the invention, the exogenous sequence replacesthe function of a defective gene in the target cell. There are severalthousand inherited genetic diseases of mammals, including humans, whichare caused by defective genes. Examples of such genetic diseases includecystic fibrosis, where there is known to be a mutation in the CFTR gene;Duchenne muscular dystrophy, where there is known to be a mutation inthe dystrophin gene; sickle cell disease, where there is known to be amutation in the HbA gene. Many types of cancer are caused by defectivegenes, especially protooncogenes, and tumour-suppressor genes that haveundergone mutation. Examples of protooncogenes are ras, src, bcl and soon; examples of tumour-suppressor genes are p53 and Rb.

In a further embodiment of the invention, the exogenous sequence encodesa Tumor Associated Antigen (TAA). TAA refers to a molecule that isdetected at a higher frequency or density in tumor cells than innon-tumor cells of the same tissue type. Examples of TAA includes butare not limited to CEA, MART1, MAGE1, MAGES, GP-100, MUC1 (see WO92/07000, WO 95/09241 and Rochlitz et al. J Gene Med. 2003August;5(8):690-9 incorporated herein by reference), MUC2, pointedmutated ras oncogene, normal or point mutated p53, overexpressed p53,CA-125, PSA, C-erb/B2, BRCA I, BRCA II, PSMA, tyrosinase, TRP1, TRP2,NY-ESO-1, TAG72, KSA, HER-2/neu, bcr-abl, pax3-fkhr, ews-fli-1,surviving and LRP. According to a more preferred embodiment the TAA isMUC1.

In another embodiment of the invention, the exogenous gene encodes anantigen. As used herein, “antigen” refers to a ligand that can be boundby an antibody; an antigen need not itself be immunogenic. Preferablythe antigen is derived from a virus such as for example HIV-1, (such asgp 120 or gp 160), any of Feline Immunodeficiency virus, human or animalherpes viruses, such as gD or derivatives thereof or Immediate Earlyprotein such as ICP27 from HSV1 or HSV2, cytomegalovirus (such as gB orderivatives thereof), Varicella Zoster Virus (such as gpl, II or III),or from a hepatitis virus such as hepatitis B virus (HBV) for exampleHepatitis B Surface antigen or a derivative thereof, hepatitis A virus(HAV), hepatitis C virus (HCV; see WO 04/111082; preferentially nonstructural HCV protein from genotype 1b strain ja), and hepatitis Evirus (HEV), or from other viral pathogens, such as RespiratorySyncytial Virus, Human Papilloma Virus (HPV; see WO 90/10459, WO95/09241, WO 98/04705, WO 99/03885 WO 07/121894 and WO 07/121894; E6 andE7 protein from the HPV16 strain are preferred; see also Liu et al. ProcNatl Acad Sci U S A. 2004 Oct. 5;101 Suppl 2:14567-71) or Influenzavirus, or derived from bacterial pathogens such as Salmonella,Neisseria, Borrelia (for example OspA or OspB or derivatives thereof),or Chlamydia, or Bordetella for example P.69, PT and FHA, or derivedfrom parasites such as plasmodium or Toxoplasma. According to a morepreferred embodiment the antigen is selected from HCV or HPV.

With this regard, preferred recombinant Orthopoxviruses producedaccording to the method of the invention is MVA-HCV (see WO 04/111082)also called TG4040.

The recombinant Orthopoxvirus can comprise more than one exogenoussequence and each exogenous sequence can encodes more than one molecule.For example, it can be useful to associate in a same recombinantOrthopoxvirus, an exogenous sequenced encoding e.g. a TAA (as previouslydescribed) or an antigen (as previously described) with an exogenoussequence encoding a cytokine (e.g. interleukin (IL as for instance IL2);tumour necrosis factor (TNF); interferon-(IFN); colony stimulatingfactor (CSF)).

With this regard, preferred recombinant Orthopoxviruses producedaccording to the method of the invention are:

-   -   MVA-[MUC1-1L2] (see WO 92/07000 and WO 95/09241) also called        TG4010; and    -   MVA-[HPV-IL2] (see WO 90/10459, WO 95/09241, WO 98/04705, WO        99/03885, WO 07/121894 and WO 07/121894) also called TG4001.

Advantageously, the recombinant Orthopoxvirus further comprises theelements necessary for the expression of the exogenous sequence(s). Theelements necessary for the expression comprise of the set of elementsallowing the transcription of a nucleotide sequence to RNA and thetranslation of a mRNA to a polypeptide, in particular the promotersequences and/or regulatory sequences which are effective in the cell tobe infected by the recombinant Orthopoxvirus of the invention, andoptionally the sequences required to allow the excretion or theexpression at the surface of the cells for said polypeptide. Theseelements may be inducible or constitutive. Of course, the promoter isadapted to the recombinant Orthopoxvirus selected and to the host cell.There may be mentioned, by way of example, the Vaccinia Virus promotersp7.5K pH5R, pK1L, p28, p11 or a combination of said promoters. Theliterature provides a large amount of information relating to suchpromoter sequences. The elements necessary can, in addition, includeadditional elements which improve the expression of the exogenoussequence or its maintenance in the host cell. There may be mentioned inparticular the intron sequences (WO 94/29471), secretion signalsequences, nuclear localization sequences, internal sites forreinitiation of translation of the IRES type, poly A sequences fortermination of transcription.

The present invention also relates to a purified wild type, attenuatedand/or recombinant Orthopoxvirus obtained by the method of the presentinvention for use as a pharmaceutical composition, preferably as avaccine.

As used herein, a “pharmaceutical composition” refers to a compositioncomprising a pharmaceutically acceptable carrier. Said pharmaceuticallyacceptable carrier is preferably isotonic, hypotonic or weaklyhypertonic and has a relatively low ionic strength, such as for examplea sucrose solution. Moreover, such a carrier may contain any solvent, oraqueous or partially aqueous liquid such as nonpyrogenic sterile water.The pH of the pharmaceutical composition is, in addition, adjusted andbuffered so as to meet the requirements of use in vivo. Thepharmaceutical compositions may also include a pharmaceuticallyacceptable diluent, adjuvant or excipient, as well as solubilizing,stabilizing and preserving agents. For injectable administration, aformulation in aqueous, nonaqueous or isotonic solution is preferred. Itmay be provided in a single dose or in a multidose in liquid or dry(powder, lyophilisate and the like) form which can be reconstituted atthe time of use with an appropriate diluent.

The present invention also relates to a purified wild type, attenuatedand/or recombinant Orthopoxvirus obtained by the method of the presentinvention for the treatment and/or the prevention a cancer, aninfectious disease and/or an autoimmune disorder.

As used herein, “cancer” refers but is not limited to lung cancer (e.g.small cell lung carcinomas and non-small cell lung), bronchial cancer,oesophageal cancer, pharyngeal cancer, head and neck cancer (e.g.laryngeal cancer, lip cancer, nasal cavity and paranasal sinus cancerand throat cancer), oral cavity cancer (e.g. tongue cancer), gastriccancer (e.g. stomach cancer), intestinal cancer, gastrointestinalcancer, colon cancer, rectal cancer, colorectal cancer, anal cancer,liver cancer, pancreatic cancer, urinary tract cancer, bladder cancer,thyroid cancer, kidney cancer, carcinoma, adenocarcinoma, skin cancer(e.g. melanoma), eye cancer (e.g. retinoblastoma), brain cancer (e.g.glioma, medulloblastoma and cerebral astrocytoma), central nervoussystem cancer, lymphoma (e.g. cutaneous B-cell lymphoma, Burkitt'slymphoma, Hodgkin's syndrome and non-Hodgkin's lymphoma), bone cancer,leukaemia, breast cancer, genital tract cancer, cervical cancer (e.g.cervical intraepithelial neoplasia), uterine cancer (e.g. endometrialcancer), ovarian cancer, vaginal cancer, vulvar cancer, prostate cancer,testicular cancer. “Cancers” also refer to virus-induced tumors,including, but is not limited to papilloma virus-induced carcinoma,herpes virus-induced tumors, EBV-induced B-cell lymphoma, hepatitisB-induced tumors, HTLV-1-induced lymphoma and HTLV-2-induced lymphoma.

As used herein, “infectious disease” refers to any disease that iscaused by an infectious organism. Infectious organisms include, but arenot limited to, viruses (e.g. single stranded RNA viruses, singlestranded DNA viruses, human immunodeficiency virus (HIV), hepatitis A,B, and C virus, herpes simplex virus (HSV), cytomegalovirus (CMV),respiratory syncytial virus (RSV), Epstein-Barr virus (EBV) or humanpapilloma virus (HPV)), parasites (e.g. protozoan and metazoan pathogenssuch as Plasmodia species, Leishmania species, Schistosoma species orTrypanosoma species), bacteria (e.g. Mycobacteria in particular, M.tuberculosis, Salmonella, Streptococci, E. coli or Staphylococci), fungi(e.g. Candida species or Aspergillus species), Pneumocystis Carinii, andprions.

As used herein, “autoimmune disorder” refers to two general types:‘Systemic autoimmune diseases’ (i.e., disorders that damage many organsor tissues), and ‘localized autoimmune diseases’ (i.e., disorders thatdamage only a single organ or tissue). However, the effect of ‘localizedautoimmune diseases’, can be systemic by indirectly affecting other bodyorgans and systems. ‘Systemic autoimmune diseases’ include but are notlimited to rheumatoid arthritis which can affect joints, and possiblylung and skin; lupus, including systemic lupus erythematosus (SLE),which can affect skin, joints, kidneys, heart, brain, red blood cells,as well as other tissues and organs; scleroderma, which can affect skin,intestine, and lungs; Sjogren's syndrome, which can affect salivaryglands, tear glands, and joints; Goodpasture's syndrome, which canaffect lungs and kidneys; Wegener's granulomatosis, which can affectsinuses, lungs, and kidneys; polymyalgia rheumatica, which can affectlarge muscle groups, and temporal arteritis/giant cell arteritis, whichcan affect arteries of the head and neck. ‘Localized autoimmunediseases’ include but are not limited to Type 1 Diabetes Mellitus, whichaffects pancreas islets; Hashimoto's thyroiditis and Graves' disease,which affect the thyroid; celiac disease, Crohn's diseases, andulcerative colitis, which affect the gastrointestinal tract; multiplesclerosis (MS) and Guillain-Barre syndrome, which affect the centralnervous system; Addison's disease, which affects the adrenal glands;primary biliary sclerosis, sclerosing cholangitis, and autoimmunehepatitis, which affect the liver; and Raynaud's phenomenon, which canaffect the fingers, toes, nose, ears.

The present invention also relates to a pharmaceutical composition,preferably a vaccine, comprising a purified wild type, attenuated and/orrecombinant Orthopoxvirus obtained by the method of the presentinvention. According to the invention, said pharmaceutical compositionis intended for the treatment and/or the prevention a cancer, aninfectious disease and/or an autoimmune disorder.

The present invention also relates to the use of a purified wild type,attenuated and/or recombinant Orthopoxvirus obtained by the method ofthe present invention for the preparation of a pharmaceuticalcomposition, preferably a vaccine, for the treatment and/or theprevention a cancer, an infectious disease and/or an autoimmunedisorder.

The pharmaceutical composition and in particular the vaccine may bemanufactured conventionally for administration by the local, parenteralor digestive route. The routes of administration may be for instance theintragastric, subcutaneous, intracardiac, intramuscular, intravenous,intraperitoneal, intratumor, intranasal, intrapulmonary or intratrachealroute. For the latter three embodiments, administration by aerosol orinstillation is advantageous. The administration may be made as a singledose or repeated once or several times after a certain time interval.The appropriate route of administration and dosage vary as a function ofvarious parameters, for example, of the individual, of the disease to betreated or of the gene(s) of interest to be transferred. According to afirst possibility, the pharmaceutical composition and in particular thevaccine may be administered directly in vivo (for example by intravenousinjection, into an accessible tumor or at its periphery, subcutaneouslyfor a therapeutic or prophylactic vaccination). It is also possible toadopt the ex vivo approach which consists in collecting cells from thepatient (bone marrow stem cells, peripheral blood lymphocytes, musclecells and the like), transfecting or infecting them in vitro accordingto prior art techniques and readministering them to the patient. It ismoreover possible to envisage, where appropriate and without departingfrom the scope of the present invention, carrying out simultaneous orsuccessive administrations, by different routes, of the variouscomponents contained in the pharmaceutical composition and in particularin the vaccine.

The present invention also relates to the use of an immortalized aviancell line obtained from an avian cell belonging to the Anatidae familyfor the production of a wild type, attenuated and/or recombinantOrthopoxvirus according to the method of the invention. Among Anatidae,cell belonging to the Cairina or Anas genus is particularly preferred.Even more preferably, the immortalized avian cell lines belong to theCairina moschata or to the Anas platyrhynchos species.

According to a preferred embodiment of the invention, Cairina moschataimmortalized avian cell lines are Cairina moschata immortalized aviancell lines comprising a nucleic acid sequence coding a telomerasereverse transcriptase (TERT) covered by patent application WO2007/077256. Are particularly preferred, the following immortalizedavian cell lines:

-   -   T3-17490 as deposited at the European Collection of Cell        Cultures (ECACC) under accession number 08060502 (see FIGS. 2, 3        and 4) or a derivative thereof;    -   T6-17490 as deposited at the European Collection of Cell        Cultures (ECACC) under accession number 08060501 (see FIGS. 5, 6        and 7) or a derivative thereof.

With this regard, the present invention also relates to:

-   -   The use of a Cairina moschata immortalized avian cell line        comprising a nucleic acid sequence coding a telomerase reverse        transcriptase (TERT) for the production of a wild type,        attenuated and/or recombinant Orthopoxvirus according to the        method of the invention.    -   The use of T3-17490 Cairina moschata immortalized avian cell        line as deposited at the European Collection of Cell Cultures        (ECACC) under accession number 08060502 (as described in        Example 2) or a derivative thereof for the production of a wild        type, attenuated and/or recombinant Orthopoxvirus according to        the method of the invention.    -   The use of T6-17490 Cairina moschata immortalized avian cell        line as deposited at the European Collection of Cell Cultures        (ECACC) under accession number 08060501 (as described in        Example 3) or a derivative thereof for the production of a wild        type, attenuated and/or recombinant Orthopoxvirus according to        the method of the invention.

According to another preferred embodiment of the invention, Cairinamoschata immortalized avian cell lines are Cairina moschata immortalizedavian cell lines comprising an E1A nucleic acid sequence and a nucleicacid sequence coding a telomerase reverse transcriptase (TERT) coveredby patent application WO 2009/004016.

With this regard, the present invention also relates to the use of aCairina moschata immortalized avian cell line comprising an E1A nucleicacid sequence and a nucleic acid sequence coding a telomerase reversetranscriptase (TERT) for the production of a wild type, attenuatedand/or recombinant Orthopoxvirus according to the method of theinvention.

As used throughout the entire application, “derivative” of the depositedimmortalized avian cell lines refers to an immortalized avian cell linewhich comprises a nucleic acid sequence coding a “substance ofinterest”. As used herein, a “substance of interest” can include, but isnot limited to, a pharmaceutically active protein e.g. growth factors,growth regulators, antibodies, antigens, their derivatives useful forimmunization or vaccination and the like, interleukins, insulin,erythropoietin, G-CSF, GM-CSF, hPG-CSF, M-CSF, interferons(interferon-alpha, interferon-beta, interferon-gamma), blood clottingfactors (e.g. Factor VIII; Factor IX; tPA) or combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: depicts the effect of various Benzonase® concentrations (i.e. 10mM; 50 mM) on Benzonase® endonuclease activity (temperature of 25° C.;Mg²⁺ 2 mM; pH 8).

FIG. 2: depicts light microscopy imaging of T3-17490 (ECACC 08060502)Cairina moschata immortalized avian cell line (passage 39).

FIG. 3: despicts T3-17490 (ECACC 08060502) Cairina moschata immortalizedavian cell line growth curve (from passage 7 to passage 75).

FIG. 4: depicts T3-17490 (ECACC 08060502) Cairina moschata immortalizedavian cell line population doubling time evolution (from passage 7 topassage 75).

FIG. 5: depicts light microscopy imaging of T6-17490 (ECACC 08060501)(passage 45).

FIG. 6: T6-17490 (ECACC 08060501) Cairina moschata immortalized aviancell line growth curve (from passage 15 to passage 51).

FIG. 7: T6-17490 (ECACC 08060501) Cairina moschata immortalized aviancell line population doubling time evolution (from passage 16 to passage51).

To illustrate the invention, the following examples are provided. Theexamples are not intended to limit the scope of the invention in anyway.

EXAMPLES Example 1 Methode A Step a): Preparing a Culture of PackagingCells.

Sixty six SPF eggs are incubated in for 60 secondes in a 2% formolsolution. After being rinsed with 70% ethanol, the eggs are opened, theembryos are extracted and dissected. The obtained tissues are thendigested at 36.5° C. for 120 minutes by dispase (UI/ml) and tripleselect (UI/ml). The mixture is filtrated to remove undigested tissuesand the CEFs are collected by centrifugation (2300 rpm, 15 minutes). TheCEFs are incubated in 55 L of VP-SFM (Invitrogen) for 2 days at 36.5° C.

Step b): Infecting the Packaging Cell Culture with an Orthopoxvirus.

The cell culture media is then discarded and the MVA-[MUC1-IL2] alsocalled TG4010 (0.05 MOI) (MVA deposited before Collection Nationale deCultures de Microorganismes (CNCM) under depositary N° I-721) is addedin 55 L of Basal Medium Eagle (Invitrogen).

Step c): Culturing the Infected Packaging Cells until ProgenyOrthopoxvirus is Produced.

The infected CEFs are then incubated for three days at 36.5° C.

Step d): Incubation in Presence of One or More Nucleases.

Infected CEFs comprising the MVA progeny are then incubated in presenceof Benzonase® 10 U/ml or 50 U/ml (Merck; Reference 1.01653.0001) underthe following conditions:

-   -   2 hours under agitation at a temperature of 25° C.;    -   Mg²⁺ 2 mM;    -   pH of 8.0.        Step e): Recovering the Orthopoxviruses from the Culture        Supernatant and/or the Packaging Cells.

The cell culture media and the CEFs are collected. The mixture is thenhomogenised for 15 minutes at 11 ml/min with a Silverson© L4R high speedhomogeniser (Silverson), or for 75 minutes at 1500 ml/min with aSONITUBE 36 kHz type SM 35/3WU (Heraeus PSP).

The obtained mixture is then incubated 2 hours under agitation at atemperature of 25° C. and pH 8.0.

Step f): Adding Monovalent Salts to the Orthopoxviruses Recovered inStep e) Under Suitable Conditions to Inhibit the Nuclease(s) Activityand to Avoid the Adsorption of Said Orthopoxviruses to the AnionExchange Adsorbent in Step g).

The obtained mixture is then incubated in presence of NaCl 250 mM and pH8.0.

Step g): Contacting the Mixture Obtained in Step f) with an AnionExchange Adsorbent Under Suitable Conditions to Allow the Capture ofNucleic Acids.

The obtained mixture is then added to UNOsphere® Q (BioRad). TheUNOsphere® Q (BioRad) beads-formed matrix is first washed with sterilewater, then autoclavated in Tris 10 mM buffer (pH 8.0), and thenequilibrated with NaCl 250 mM or 300 mM buffer (pH 8.0).

The UNOsphere® Q (BioRad) beads-formed matrix is then added by using aperistaltic Watson-Marlow pump (Reference 323ES/4D, 520S; Watson-Marlow)to the mixture obtained in step f) contained in a Flexboy® bag(Reference FFB101961; Sartorius Stedim biotech).

The UNOsphere® Q (BioRad) beads-formed matrix and the mixture obtainedin step f) are kept in contact during 1 hour under slow agitation atroom temperature (20° C. to 22° C.).

Step h): Clarifying the Mixture Obtained in Step g) Under SuitableConditions to Allow the Withdrawal of the Cellular Debris.

The obtained mixture is then clarified by depth filtration on aSartopure® PP2 8 μm (Sartorius) coupled to a Sartopure® PP2 5 μm(Sartorius) at a flow rate of 1 L/minute.

Step i): Washing of the Anion Exchange Adsorbent with a SolutionComprising Monovalent Salts Under Suitable Conditions to Recover theRemained Orthopoxviruses in the Flow Through.

The UNOsphere® Q (BioRad) is then washed (v/v) with NaCl 250 mM or 300mM in SO8 buffer (10 mM Tris-HCl; sucrose 5% (w/v); 10 mM sodiumglutamate; 50 mM NaCl; pH 8.0 with physiological osmolarity (290mOsm/kg)) by using a peristaltic Watson-Marlow pump (Reference 323ES/4D,520S; Watson-Marlow).

The flow trough obtained in step h) and the flow trough obtained in stepi) are then incubated overnight (i.e. 18 hours) at 5° C. in presence ofsaccharose 50 g/L final.

Step j): Concentrating the Flow Through Obtained in Step h) and the FlowThrough Obtained in Step i).

The flow trough obtained in step h) and the flow trough obtained in stepi) are then concentrated 18 times through a 0.1 μm ProstakMicrofiltration Module (Reference PSVVAG021, Millipore).

Step k): Diafiltrating the Fraction Comprising the OrthopoxvirusesObtained in Step j).

The retentate is then diafiltred on the same module i.e. 0.1 μm ProstakMicrofiltration Module (Reference PSVVAG021, Millipore).

The quantification of DNA is performed using Quant-iT™ Picogreen® dsDNAAssays Kit (Cat. No. P7589, Invitrogen).

Results: No remaining DNA is detected.

Example 2 T3-17490 Immortalized Cairina Moschata Cell Line (as Depositedat ECACC Under Accession Number 08060502) Used for the Production of anOrthopoxvirus According to the Method of the Invention

T3-17490 cells (passage 39) have a homogenous fibroblast like morphology(FIG. 2). The static monolayer is stable up to 100% confluence andsubject to contact inhibition. The cells were tested negative formycoplasma contamination and for microbial contamination as well. TheT3-17490 cell line growth curve (from passage 7 to passage 75) (FIG. 3)shows a continuous exponential growth phase from passage 19 to passage75. Focusing on the evolution of the population doubling time (PDT), aprogressive stabilisation and decrease is observed, in particular it canbe noted that the PDT is stabilised under the 48 h mark during the 10latest passages (FIG. 4). The corresponding number of populationdoublings (population doubling level, PDL) has been calculated bycumulating the 2 exponential growth phases: during the 75 passages thecells have undergone at least 147 population doublings (PD). The numberof population doublings a primary cell can undergo before enteringsenescence is tissue and specie dependent. It is commonly admitted thatthe upper limit is situated between 50 and 60 PD. The T3-17490 cells aretherefore far beyond the Hayflick limit and are consequently referred asimmortalized cell line.

N.B.:

-   -   The population doubling level (PDL) refers to the number of cell        generations (biomass 2 fold increase). PDL calculation:        PDL=Ln(final/initial cell number)/Ln(2);    -   The population doubling time (PDT), also called generation time,        is the time needed for one population doubling. PDT calculation:        PDT=Δt*Ln(2)/Ln(final/initial cell number).

Example 3 T6-17490 Immortalized Cairina Moschata Cell Line (as Depositedat ECACC Under Accession Number 08060501) Used for the Production of anOrthopoxvirus According to the Method of the Invention

T6-17490 cells (passage 45) have a homogenous fibroblast like morphology(FIG. 5). The static monolayer is stable up to 100% confluence andsubject to contact inhibition. The cells were tested negative formycoplasma contamination and for microbial contamination as well. TheT6-17490 cell line growth curve (from passage 15 to passage 51) (FIG. 6)shows a continuous exponential growth phase from passage 19. During thisperiod the measured population doubling time (PDT) was progressivelydecreasing. Average PDT passed from 94 h (passage 20 to 35) to 52 h(passage 36 to 51) (FIG. 7). The number of calculated populationdoublings (PDL) corresponding to the 51 passages is at least 71population doublings. The T6-17490 cells are therefore far beyond theHayflick limit and are consequently referred as immortalized cell line.

N.B.:

-   -   The population doubling level (PDL) refers to the number of cell        generations (biomass 2 fold increase). PDL calculation:        PDL=Ln(final/initial cell number)/Ln(2);    -   The population doubling time (PDT), also called generation time,        is the time needed for one population doubling. PDT calculation:        PDT=Δt* Ln(2)/Ln(final/initial cell number).

Example 4 Methode B

Step a′): Preparing a Culture of Packaging Cells.

Sixty six SPF eggs are incubated in for 60 secondes in a 2% formolsolution. After being rinsed with 70% ethanol, the eggs are opened, theembryos are extracted and dissected. The obtained tissues are thendigested at 36.5° C. for 120 minutes by dispase (UI/ml) and tripleselect (UI/ml). The mixture is filtrated to remove undigested tissuesand the CEFs are collected by centrifugation (2300 rpm, 15 minutes). TheCEFs are incubated in 55 L of VP-SFM (Invitrogen) for 2 days at 36.5° C.

Step b′): Infecting the Packaging Cell Culture with an Orthopoxvirus.

The cell culture media is then discarded and the MVA-[MUC1-IL2] alsocalled TG4010 (0.05 MOI) (MVA deposited before Collection Nationale deCultures de Microorganismes (CNCM) under depositary N° I-721) is addedin 55 L of Basal Medium Eagle (Invitrogen).

Step c′): Culturing the Infected Packaging Cells Until ProgenyOrthopoxvirus is Produced.

The infected CEFs are then incubated for three days at 36.5° C.

Step d′): Incubation in Presence of One or More Nucleases.

Infected CEFs comprising the MVA progeny are then incubated in presenceof Benzonase® 10 U/ml (Merck; Reference 1.01653.0001) under thefollowing conditions:

-   -   2 hours under agitation at a temperature of 25° C.;    -   Mg²⁺ 2 mM;    -   pH of 8.0.        Step e′): Recovering the Orthopoxviruses from the Culture        Supernatant and/or the Packaging Cells.

The cell culture media and the CEFs are collected. The mixture is thenhomogenised for 15 minutes at 11 ml/min with a Silverson© L4R high speedhomogeriiser (Silverson), or for 75 minutes at 1500 ml/min with aSONITUBE 36 kHz type SM 35/3WU (Heraeus PSP).

Step f′): Incubating the Orthopoxviruses Recovered in Step e′) inPresence of One or More Agents Capable to Inhibit the Nuclease(s)Activity.

The obtained mixture is then incubated in presence of NaCl 100 mM andEDTA 10 mM, pH 8.0.

Step g′): Contacting the Mixture Obtained in Step f′) with an AnionExchange Adsorbent Under Suitable Conditions to Allow the Capture ofsaid Orthopoxviruses and Nucleic Acids.

The obtained mixture is then added to BioSepra® Q hyperZ (PallCorporation). The BioSepra® hyperZ (Pall Corporation) beads-formedmatrix is first washed with sterile water, then sanitized with NaOH 0,5N, washed with Tris 10 mM buffer (pH 8.0), and then equilibrated withTris 10 mM NaCl 10 mM saccharose 5% buffer (pH 8.0).

The BioSepra® Q hyperZ (Pall Corporation) beads-formed matrix is thenadded by using a peristaltic Watson-Marlow pump (Reference 323ES/4D,520S; Watson-Marlow) to the mixture obtained in step f′) contained in aFlexboy® bag (Reference FFB101961; Sartorius Stedim biotech).

The BioSepra® Q hyperZ (Pall Corporation) beads-formed matrix and themixture obtained in step f′) are kept in contact during 1 hour underslow agitation at room temperature (20° C. to 22° C.).

Step h′): Clarifying the Mixture Obtained in Step g′) Under SuitableConditions to Allow the Withdrawal of the Cellular Debris.

The obtained mixture is then clarified by depth filtration on aSartopure® PP2 8 μm (Sartorius) coupled to a Sartopure® PP2 5 μm(Sartorius) at a flow rate of 1 L/minute.

Step i′): Eluting the Orthopoxviruses with a Solution ComprisingMonovalent Salts.

The Orthopoxviruses are eluted by an increasing NaCl concentrationgradient (300 mM; 400 mM; 500 mM) in SO8 buffer (10 mM Tris-HCl; sucrose5% (w/v); 10 mM sodium glutamate; 50 mM NaCl; pH 8.0 with physiologicalosmolarity (290 mOsm/kg)) by using a peristaltic Watson-Marlow pump(Reference 323ES/4D, 520S; Watson-Marlow).

Step j′): Concentrating the Mixture Obtained in Step i′).

The eluat obtained in step i′) is then concentrated 18 times through a0.1 μm Prostak Microfiltration Module (Reference PSVVAG021, Millipore).

Step k′): Diafiltrating the Fraction Comprising the OrthopoxvirusesObtained in Step j′).

The retentate is then diafiltred on the same module i.e. 0.1 μm ProstakMicrofiltration Module (Reference PSVVAG021, Millipore).

The quantification of DNA is performed using Quant-iT™ Picogreen® dsDNAAssays Kit (Cat. No. P7589, Invitrogen).

Results: No remaining DNA is detected.

All documents (e.g. patents, patent applications, publications) cited inthe above specification are herein incorporated by reference. Variousmodifications and variations of the present invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention which are obvious to those skilledin the art are intended to be within the scope of the following claims.

1-42. (canceled)
 43. A method for producing and purifying anOrthopoxvirus, comprising: a) preparing a culture of packaging cells; b)infecting the packaging cell culture with an Orthopoxvirus; c) culturingthe infected packaging cells until progeny Orthopoxvirus is produced; d)incubating the cultured infected packaging cells in the presence of oneor more nucleases; e) recovering Orthopoxviruses from the culturesupernatant and/or the packaging cells; f) adding monovalent salts tothe Orthopoxviruses recovered in step e) under conditions suitable toinhibit the nuclease(s) activity and to avoid the adsorption of saidOrthopoxviruses to an anion exchange adsorbent; and, g) contacting themixture obtained in step f) with said anion exchange adsorbent underconditions suitable to allow the capture of nucleic acids.
 44. Themethod according to claim 43, wherein the packaging cells areimmortalized cell lines.
 45. The method according to claim 44, whereinthe packaging cells are immortalized avian cell lines.
 46. The methodaccording to claim 44, wherein said immortalized cell lines are capableof growing in suspension without microcarriers.
 47. The method accordingto claim 46, wherein said immortalized cell lines are capable of growingin suspension without microcarriers and wherein said cells are culturedin media free from animal products.
 48. The method according to claim43, wherein the packaging cells are primary or secondary avian cells.49. The method according to claim 43, wherein the packaging cells arechicken embryo fibroblasts (CEFs).
 50. The method according to claim 43,wherein the pH for carrying out step d) is between 7.0 and 9.0.
 51. Themethod according to claim 50, wherein the pH is between 7.5 and 8.5. 52.The method according to claim 51, wherein the pH is 8.0.
 53. The methodaccording to claim 43, wherein the nuclease(s) is/are endonuclease(s).54. The method according to claim 43, wherein the concentration ofnuclease(s) is in the range of 5 to 100 U/ml.
 55. The method accordingto claim 54, wherein the concentration of nuclease(s) is in the range of5 to 50 U/ml.
 56. The method according to claim 55, wherein theconcentration of nuclease(s) is 10 U/ml.
 57. The method according toclaim 43, wherein the step of recovering the Orthopoxviruses is precededby: 1) a step allowing the disruption of the packaging cell membrane;and 2) a step of incubation of the mixture obtained in step 1) for least1 hour with the one or more nuclease(s) of step (d).
 58. The methodaccording to claim 43, wherein the anion exchange adsorbent is abeads-formed matrix having a diameter higher than 8 μm.
 59. The methodaccording to claim 58, wherein the anion exchange adsorbent consists ina beads-formed matrix having a diameter between 50 μm and 150 μm. 60.The method according to claim 59, wherein the anion exchange adsorbentis a beads-formed matrix having a diameter between 90 μm and 120 μm. 61.The method according to claim 60, wherein the anion exchange adsorbentis a beads-formed matrix having a diameter of 120 μm.
 62. The methodaccording to claim 43, wherein the functional groups of the anionexchange adsorbent are selected from the group consisting ofdimethylaminoethyl (DMAE), diethylaminoethyl (DEAE), trimethylaminoethyl(TMAE), and triethylaminoethyl (TEAE).
 63. The method according to claim62, wherein the functional groups of the anion exchange adsorbent aretrimethylaminoethyl (TMAE).
 64. The method according to claim 43,wherein the monovalent salts used in step f) are NaCl.
 65. The methodaccording to claim 64, wherein the concentration of monovalent saltsused in step f) is 50 to 150 mM.
 66. The method according to claim 65,wherein the concentration of monovalent salts used in step f) is 100 mM.67. The method according to claim 43, further comprising one or moreadditional purification steps.
 68. The method according to claim 67,further comprising: h) optionally clarifying the mixture obtained instep g) under suitable conditions to allow the withdrawal of thecellular debris; i) washing the anion exchange adsorbent with a solutioncomprising monovalent salts under suitable conditions to recoverOrthopoxviruses in the flow through; and, j) concentrating the flowthrough obtained in step g) or step h) and the flow through obtained instep i).
 69. The method according to claim 68, wherein the clarificationstep is performed by depth filtration.
 70. The method according to claim68, wherein the clarification step is performed by depth filtration withfilters having a pore size of 8 μm coupled to filters having a pore sizeof 5 μm.
 71. The method according to claim 68, wherein the concentrationstep is performed by microfiltration with filters having a pore sizebetween 0.09 and 0.15 μm.
 72. The method according to claim 71, whereinthe concentration step is performed by microfiltration with filtershaving a pore size of 0.1 μm.
 73. The method according to claim 68,further comprising a diafiltration step performed with filters having apore size between 0.09 and 0.15 μm.
 74. The method according to claim68, further comprising a diafiltration step performed over filtershaving a pore size of 0.1 μm.
 75. The method according to claim 68,wherein the method further comprises: 1) a step of gel filtration; and2) a step of diafiltration.
 76. The method according to claim 43,wherein said Orthopoxvirus is a Vaccinia Virus or a modified VacciniaVirus Ankara (MVA).
 77. The method according to claim 67, wherein saidOrthopoxvirus is a Vaccinia Virus or a modified Vaccinia Virus Ankara(MVA).
 78. The method according to claim 68, wherein said Orthopoxvirusis a Vaccinia Virus or a modified Vaccinia Virus Ankara (MVA).
 79. Themethod according to claim 75, wherein said Orthopoxvirus is a VacciniaVirus or a modified Vaccinia Virus Ankara (MVA).
 80. A method forproducing and purifying an Orthopoxvirus, comprising recoveringOrthopoxviruses from cultured packaging cells infected with theOrthopoxvirus and/or a culture supernatant of such cultured packagingcells; the improvement comprising: i) incubating the cultured infectedpackaging cells in the presence of one or more nucleases beforerecovering said Orthopoxviruses; ii) adding monovalent salts to therecovered Orthopoxviruses under conditions suitable to inhibit saidnuclease(s) and to avoid the adsorption of said Orthopoxviruses to ananion exchange adsorbent; and, iii) contacting the mixture obtained instep ii) with said anion exchange adsorbent under conditions suitable toallow the capture of nucleic acids.
 81. The method according to claim80, wherein the packaging cells are immortalized cell lines.
 82. Themethod according to claim 80, wherein the packaging cells areimmortalized avian cell lines.
 83. The method according to claim 81,wherein said immortalized cell lines are capable of growing insuspension without microcarriers.
 84. The method according to claim 83,wherein said immortalized cell lines are capable of growing insuspension without microcarriers and wherein said cells are cultured inmedia free from animal products.
 85. The method according to claim 80,wherein the packaging cells are primary or secondary avian cells. 86.The method according to claim 80, wherein the packaging cells arechicken embryo fibroblasts (CEFs).
 87. The method according to claim 80,wherein the pH for carrying out step d) is between 7.0 and 9.0.
 88. Themethod according to claim 87, wherein the pH is between 7.5 and 8.5. 89.The method according to claim 88, wherein the pH is 8.0.
 90. The methodaccording to claim 80, wherein the nuclease(s) is/are endonuclease(s).91. The method according to claim 80, wherein the concentration ofnuclease(s) is in the range of 5 to 100 U/ml.
 92. The method accordingto claim 91, wherein the concentration of nuclease(s) is in the range of5 to 50 U/ml.
 93. The method according to claim 92, wherein theconcentration of nuclease(s) is 10 U/ml.
 94. The method according toclaim 80, wherein the anion exchange adsorbent is a beads-formed matrixhaving a diameter higher than 8 μm.
 95. The method according to claim94, wherein the anion exchange adsorbent consists in a beads-formedmatrix having a diameter between 50 μm and 150 μm.
 96. The methodaccording to claim 95, wherein the anion exchange adsorbent is abeads-formed matrix having a diameter between 90 μm and 120 μm.
 97. Themethod according to claim 96, wherein the anion exchange adsorbent is abeads-formed matrix having a diameter of 120 μm.
 98. The methodaccording to claim 80, wherein the functional groups of the anionexchange adsorbent are selected from the group consisting ofdimethylaminoethyl (DMAE), diethylaminoethyl (DEAE), trimethylaminoethyl(TMAE), and triethylaminoethyl (TEAE).
 99. The method according to claim98, wherein the functional groups of the anion exchange adsorbent aretrimethylaminoethyl (TMAE).
 100. The method according to claim 80,wherein the monovalent salts used in step f) are NaCl.
 101. The methodaccording to claim 100, wherein the concentration of monovalent saltsused in step f) is 50 to 150 mM.
 102. The method according to claim 101,wherein the concentration of monovalent salts used in step f) is 100 mM.103. The method according to claim 80, further comprising one or moreadditional purification steps.
 104. The method according to claim 103,further comprising: iv) optionally clarifying the mixture obtained instep iii) under suitable conditions to allow the withdrawal of thecellular debris; v) washing the anion exchange adsorbent with a solutioncomprising monovalent salts under suitable conditions to recoverOrthopoxviruses in the flow through; and, vi) concentrating the flowthrough obtained in step iii) or step iv) and the flow through obtainedin step v).
 105. The method according to claim 104, wherein theclarification step is performed by depth filtration.
 106. The methodaccording to claim 105, wherein the clarification step is performed bydepth filtration with filters having a pore size of 8 μm coupled tofilters having a pore size of 5 μm.
 107. The method according to claim104, wherein the concentration step is performed by microfiltration withfilters having a pore size between 0.09 and 0.15 μm.
 108. The methodaccording to claim 107, wherein the concentration step is performed bymicrofiltration with filters having a pore size of 0.1 μm.
 109. Themethod according to claim 104, further comprising a diafiltration stepperformed with filters having a pore size between 0.09 and 0.15 μm. 110.The method according to claim 109, further comprising a diafiltrationstep performed over filters having a pore size of 0.1 μm.
 111. Themethod according to claim 80, wherein said Orthopoxvirus is a VacciniaVirus or a modified Vaccinia Virus Ankara (MVA).
 112. The methodaccording to claim 103, wherein said Orthopoxvirus is a Vaccinia Virusor a modified Vaccinia Virus Ankara (MVA).
 113. The method according toclaim 104, wherein said Orthopoxvirus is a Vaccinia Virus or a modifiedVaccinia Virus Ankara (MVA).